Antibody Variants Having Modifications In The Constant Region

ABSTRACT

The present invention relates to positions in the constant region of antibodies, in particular the CH3 region of IgG4, which affect the strength of CH3-CH3 interactions. Mutations that either stabilize or destabilize this interaction are disclosed.

FIELD OF INVENTION

The present invention relates to modified antibodies that may be used intherapeutic applications. The invention also relates to methods forproducing the antibodies, pharmaceutical compositions comprising theantibodies and use thereof for different therapeutic applications.

BACKGROUND OF THE INVENTION

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each light chain is comprised of a light chainvariable region (abbreviated herein as VL) and a light chain constantregion (abbreviated herein as CL). Each heavy chain is comprised of aheavy chain variable region (VH) and a heavy chain constant region (CH)consisting of three domain, CH1, CH2 and CH3). CH1 and CH2 of the heavychain are separated from each other by the so-called hinge region. Thehinge region normally comprises one or more cysteine residues, which mayform disulphide bridges with the cysteine residues of the hinge regionof the other heavy chain in the antibody molecule.

Recently, antibodies have become a major focus area for therapeuticapplications, and many antibody drug products have been approved or arein the process of being approved for use as therapeutic drugs. Thedesired characteristics of therapeutic antibodies may vary according tothe specific condition which is to be treated. For some indications,only antigen binding is required, for instance where the therapeuticeffect of the antibody is to block interaction between the antigen andone or more specific molecules otherwise capable of binding to theantigen. For such indications, the use of Fab fragments, the onlyfunction of which is to bind antigen, may be preferred. For otherindications, further effects may also be required, such as for instancethe ability to induce complement activation and/or the ability to forinstance bind Fc receptors, protect from catabolism, recruit immunecells, etc. For such use, other parts of the antibody molecule, such asthe Fc region, may be required. Some full-length antibodies may exhibitagonistic effects (which may be considered to be undesirable) uponbinding to the target antigen, even though the antibody works as anantagonist when used as a Fab fragment. In some instances, this effectmay be attributed to “cross-linking” of the bivalent antibodies, whichin turn promotes target dimerization, which may lead to activation,especially when the target is a receptor. In the case of solubleantigens, dimerization may form undesirable immune complexes.

For some indications, monovalent antibodies may thus be preferable. Thepresently available Fab fragments show inferior pharmacokinetics due totheir small size resulting to filtration in the kidneys as well as theirinability to interact with the Brambell receptor FcRn (Junghans R P etal., Proc Natl Acad Sci USA 93(11), 5512-6 (1996)), therefore beingunstable in vivo and having very rapid clearance after administration.

There is thus a need for stable monovalent antibodies which can be usedas therapeutics.

Dimeric, monovalent antibodies (Fab/c), wherein the Fc region comprisestwo Fc polypeptides, have been described (WO200563816 to Genentech andParham P, J. Immunol. 131(6), 2895-902 (1983).

Ig half-molecules, which have a dimeric configuration consisting of onlyone light chain and only one heavy chain, have been described as theresult of rare deletions in human and murine plasmacytomas. Studies onthe biochemical nature of these half-molecules showed that they consistof IgG1 molecules in which the heavy chain CH1, hinge and CH2 regionsappeared normal, whereas deletions were found in the CH3 region. Themutations appeared to be located in CH3 and the hinge peptide appearednormal (Hobbs, J R et al., Clin Exp Immunol 5, 199 (1969); Hobbs, J R,Br Med J 2, 67 (1971); Spiegelberg, H L et al., Blood 45, 305 (1975);Spiegelberg, H L et al., Biochemistry 14, 2157 (1975); Seligmann M E etal., Ann Immunol (Paris) 129C, 855-870 (1978); Gallango, M L et al.,Blut 48, 91 (1983)). It was also showed that this human IgG1half-molecule is rapidly catabolized (half-life in man was 4.3 days)and, in monomeric form, is unable to bind C1q or Fc receptors on humanlymphocytes, monocytes or neutrophils (Spiegelberg, H L. J Clin Invest56, 588 (1975)).

Murine IgA half-molecules which were generated by somatic mutation havealso been described (Mushinski, J F, J Immunol 106, 41 (1971);Mushinski, J F et al., J Immunol 117, 1668 (1976); Potter, M et al., JMol Biol 93, 537 (1964); Robinson, E A et al., J Biol Chem 249, 6605(1974); Zack, D J et al., J Exp Med 154, 1554 (1981)). These moleculeswere shown to all contain deletions of the CH3 domain or mutations atthe CH2—CH3 boundary.

WO2007059782 (Genmab) describes human monovalent antibodies comprising alight chain and a heavy chain, wherein

a) said light chain comprises the amino acid sequence of the variable(VL) region of a selected antigen specific antibody and the amino acidsequence of the constant (CL) region of an Ig, and wherein, in case ofan IgG1 subtype, the amino sequence of the constant (CL) region has beenmodified so that it does not contain any amino acids capable ofparticipating in the formation of disulfide bonds or covalent bonds withother peptides comprising an identical amino acid sequence of theconstant (CL) region of the Ig, in the presence of polyclonal human IgGor when administered to an animal or human being, and

b) said heavy chain comprises the amino acid sequence of the variable(VH) region of said selected antigen specific antibody and the aminoacid sequence of the constant (CH) region of human Ig, wherein the aminoacid sequence of the constant (CH) region has been modified so that thehinge region and, as required by the Ig subtype, other regions of the CHregion, such as the CH3 region, does not contain any amino acid residueswhich participate in the formation of disulphide bonds or covalent orstable non-covalent inter-heavy chain bonds with other peptidescomprising an identical amino acid sequence of the constant (CN) regionof the human Ig, in the presence of polyclonal human IgG or whenadministered to an animal or human being.

As shown in WO2007059782, these monovalent antibodies have a morefavorable in vivo half-life than Fab fragments. WO2008145140 describesvariants of these monovalent antibodies wherein intermolecular CH3-CH3interactions are destabilized. The present application describesalternative and improved variants of the monovalent antibodies disclosedin WO2007059782 and WO2008145140. These variants remain monovalent evenunder conditions that favor intermolecular CH3-CH3 interactions.

Human IgG4 molecules exist in various molecular forms which differ bythe absence or presence of inter-heavy chain disulphide bonds located inthe hinge region. Thus IgG4 molecules exist in which two, one or nointer-heavy chain disulphide bonds have been formed (Schuurman, J. etal., Mol Immunol 38, 1 (2001)). Under physiological conditions, thesemolecular forms of IgG4 may be in equilibrium with each other. HumanIgG4s exist as tetramers in solution consisting of two Ig heavy and twolight chains, as common for immunoglobulin G molecules, irrespective ofthe absence or presence of these interchain disulphide bonds (Schuurman2001 supra; Gregory, L. et al. Mol Immunol 24, 821 (1987)). Only upondenaturation under non-reducing conditions, the two non-covalentlyassociated half-molecules dissociate as demonstrated bysize-determination analysis such as SDS-PAGE (Schuurman, J. et al. MolImmunol 38, 1 (2001); Deng, L. et al. Biotechnol Appl Biochem 40, 261(2004)). It has been shown that mutation of the residues of the hingeregion which are involved in inter-chain disulphide bond formation ordeletion of the hinge region lead to creation of a homogeneous pool ofIgG4 molecules in solution, which pool consists of tetrameric moleculesconsisting of two light chains and two heavy chains (Schuurman, J. etal. Mol Immunol 38, 1 (2001); Horgan, C. et al. J Immunol 150, 5400(1993)). The IgG4 hinge-deleted and mutated antibodies also demonstratedan improved capability of antigen crosslinking when compared to nativeIgG₄ molecules (Horgan, C. (1993) supra).

It has been shown that administration of two recombinant monoclonal IgG4antibodies having different antigen-binding specificities to a mouseleads to in vivo formation of bispecific antibodies. The phenomenon canbe reproduced in vitro by incubating IgG4 antibodies with cells or underreducing conditions. It has been shown that IgG4 antibodies havingdifferent antigen-binding specificities engage in Fab arm exchange whichis stochastic and in which all IgG4 molecules seem to participate. Thus,IgG4 antibodies form bispecific antibodies without concomitant formationof aggregates.

IgG4 antibodies therefore have unusual properties which are undesirablein vivo: IgG4 antibodies are unstable, dynamic, molecules which engagein Fab arm exchange. An administered therapeutic IgG4 antibody mayexchange with endogenous IgG4 antibodies with undesired specificities.The random nature of this process introduces unpredictability which ishighly undesirable for human immunotherapy.

In one aspect, the present invention relates to stabilized forms of IgG4antibodies that have a reduced ability to undergo Fab-arm exchange.Stabilized forms of IgG4 have previously been described in WO2008145142(Genmab). It has now surprisingly been found that specific alternativesubstitutions in human IgG4 can prevent Fab arm exchange, and thusstabilize IgG4.

In summary, the present invention relates to positions in the constantregion of antibodies, in particular the CH3 region of IgG4, which affectthe strength of CH3-CH3 interactions. Mutations that either stabilize ordestabilize this interaction are disclosed herein.

When introduced in the monovalent antibody context described inWO2007059782, the destabilizing mutations contribute to keeping theantibodies monovalent even under conditions that favor intermolecularCH3-CH3 interactions. When introduced in the IgG4 context, thestabilizing mutations contribute to preventing undesired Fab armexchange.

SUMMARY OF THE INVENTION

In a first main aspect, the invention relates to a monovalent antibody,which comprises

(i) a variable region of a selected antigen specific antibody or anantigen binding part of the said region, and

(ii) a CH region of an immunoglobulin or a fragment thereof comprisingthe CH2 and CH3 regions, wherein the CH region or fragment thereof hasbeen modified such that the region corresponding to the hinge regionand, if the immunoglobulin is not an IgG4 subtype, other regions of theCH region, such as the CH3 region, do not comprise any amino acidresidues which are capable of forming disulfide bonds with an identicalCH region or other covalent or stable non-covalent inter-heavy chainbonds with an identical CH region in the presence of polyclonal humanIgG,

wherein the antibody is of the IgG4 type and the constant region of theheavy chain has been modified so that one or more of the following aminoacid substitutions have been made relative to the sequence set forth inSEQ ID NO: 4: Tyr (Y) in position 217 has been replaced by Arg (R), Leu(L) in position 219 has been replaced by Asn (N) or Gln (Q), Glu (E) inposition 225 has been replaced by Thr (T), Val (V) or Ile (I), Ser (S)in position 232 has been replaced by Arg (R) or Lys (K), Thr (T) inposition 234 has been replaced by Arg (R), Lys (K) or Asn (N), Leu (L)in position 236 has been replaced by Ser (S) or Thr (T), Lys (K) inposition 238 has been replaced by Arg (R), Asp (D) in position 267 hasbeen replaced by Thr (T) or Ser (S), Phe (F) in position 273 has beenreplaced by Arg (R), Gln (Q), Lys (K) or Tyr (Y), Tyr (Y) in position275 has been replaced by Gln (Q), Lys (K) or Phe (F), Arg (R) inposition 277 has been replaced by Glu (E), Thr (T) in position 279 hasbeen replaced by Asp (D), Val (V) and Asn (N),

or the antibody is of another IgG type and the constant region of theheavy chain has been modified so that one or more of the same amino-acidsubstitutions have been made at the positions that correspond to thebefore-mentioned positions for IgG4.

As explained above, mutations at the above specified positions disfavorintermolecular CH3-CH3 interactions. Thus, monovalent antibodiescarrying these mutations are less likely to dimerize throughnon-covalent interactions. This may be an advantage for therapeuticapplications wherein such dimerization is highly undesired. Furthermore,a reduced tendency of the monovalent antibodies to associatenon-covalently through the CH3 regions may make pharmaceuticalcompositions comprising such antibodies more stable and homogenous thanpharmaceutical compositions of monovalent antibodies that do notcomprise the above-specified mutations.

Thus, in another aspect, the invention relates to a pharmaceuticalcomposition comprising the monovalent antibody according the inventionas defined herein.

In a further aspect, the invention relates to a method of treating adisease or disorder as described herein, wherein said method comprisesadministering to a subject in need of such treatment a therapeuticallyeffective amount of a monovalent antibody according to the invention.

In a further aspect, the invention relates to a stabilized IgG4 antibodyfor use as a medicament, comprising a heavy chain and a light chain,wherein said heavy chain comprises a human IgG4 constant region havingthe sequence set forth in SEQ ID NO:2, wherein Lys (K) in position 250has been replaced by Gln (Q) or Glu (E) and wherein the antibodyoptionally comprises one or more further substitutions, deletions and/orinsertions in the constant region as set forth in SEQ ID NO:2.

As explained above, and shown herein below in the Examples, themutations at position 250 stabilize the IgG4 molecule and preventundesired Fab arm exchange.

DESCRIPTION OF FIGURES

FIG. 1: Percentage of molecules present as monomers for each HG mutanttested using non-covalent nano-electrospray mass spectrometry. HG mutantsamples were prepared in aqueous 50 mM ammonium acetate solutions at aconcentration of 1 μM.

FIG. 2: NativePAGE™ Novex® Bis-Tris gel electrophoresis of CH3 mutantscompared to 2F8-HG (WT) and R277K HG mutant control.

FIG. 3: The binding of 2F8-HG and CH3 variants 2F8-HG-T234A and2F8-HG-L236V was tested in EGFR ELISA in the presence and absence ofpolyclonal human IgG.

FIG. 4: The binding of 2F8-HG and CH3 variants 2F8-HG-L236A and2F8-HG-Y275A was tested in EGFR ELISA in the presence and absence ofpolyclonal human IgG.

FIG. 5: Dose-response curves showing the inhibition of EGF-induced EGFrphosphorylation in A431 cells by anti-EGFr 2F8-HG (WT) and CH3 mutantsthereof.

FIG. 6: Percentage molecules present as monomers at different molarconcentrations of CH3 mutants compared to 2F8-HG (WT) and R277K.

FIG. 7: Relative interaction strength (KD) of CH3 mutants compared to2F8-HG (WT).

FIG. 8: The binding of 2F8-HG and deglycosylation variants 2F8-HG-GSTand 2F8-HG-NSE was tested in EGFR ELISA in the presence and absence ofpolyclonal human IgG.

FIG. 9: Percentage of molecules present as monomers for each HG mutantmeasured using non-covalent nano-electrospray mass spectrometry. HGmutant samples were prepared in aqueous 50 mM ammonium acetate solutionsat a concentration of 1 μM.

FIG. 10: Dose-response curves showing the inhibition of EGF-induced EGFrphosphorylation in A431 cells by anti-EGFr 2F8-HG (WT) andnon-glycosylation mutants thereof.

FIG. 11: Clearance (expressed as D/AUC) of non-glycosylation mutants2F8-HG-GST and 2F8-HG-NSE compared to 2F8-HG (WT) and 2F8-IgG4.

FIG. 12: Schematic representation of constructs for IgG1 and IgG4containing mutations in the core hinge and/or CH3 domain (residues arenumbered according to EU numbering, see table Example 16).

FIG. 13: Fab arm exchange of IgG1 and IgG4 hinge region or CH3 domainmutants (residues are numbered according to EU numbering, see tableExample 16).

FIG. 14: Binding of hingeless IgG4 antibody 2F8-HG and CH3 variants2F8-HG-F405L, 2F8-HG-F405A, 2F8-HG-R409A and 2F8-HG-R409K to EGFr(residues are numbered according to EU numbering, see table Example 16).Binding was tested in an EGFR ELISA in the presence and absence ofpolyclonal human IgG (IVIG).

FIG. 15: Sequence alignment of anti-EGFr antibody 2F8 in an IgG1, IgG4and (partial) IgG3 backbone. Amino acid numbering according to Kabat andaccording to the EU-index are depicted (both described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).

FIG. 16: Fab-arm exchange of CH3 domain mutants of human IgG4antibodies. Mixtures of two recombinant human IgG4 antibodies (IgG4-CD20and IgG4-EGFr) and CH3 domain mutants thereof were incubated with 0.5 mMGSH at 37° C. The formation of bispecific antibodies through Fab armexchange was followed over time and measured in a sandwich ELISA. Thebispecific activity of IgG4 at 24 hrs was set as 100%.

FIG. 17: Relative interaction strength (K_(D)) of CH3 mutants comparedto his-CH2—CH3(G4) (WT).

FIG. 18: Correlation between the CH3-CH3 interaction strength (K_(D))and the bispecific activity. The bispecific activity of IgG4 at 24 hrswas set as 100% (open circle).

DETAILED DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID No: 1: The nucleic acid sequence of the wildtype CH region ofhuman IgG4

SEQ ID No: 2: The amino acid sequence of the wildtype CH region of humanIgG4. Sequences in italics represent the CH1 region, highlightedsequences represent the hinge region, regular sequences represent theCH2 region and underlined sequences represent the CH3 region.

SEQ ID No: 3: The nucleic acid sequence of the CH region of human IgG4(SEQ ID No: 1) mutated in positions 714 and 722

SEQ ID No: 4: The amino acid sequence of the hingeless CH region of ahuman IgG4. Underlined sequences represent the CH3 region.

SEQ ID No: 5: The amino acid sequence of the lambda chain constant human(accession number S25751)

SEQ ID No: 6: The amino acid sequence of the kappa chain constant human(accession number P01834)

SEQ ID No: 7: The amino acid sequence of IgG1 constant region (accessionnumber P01857). Sequences in italics represent the CH1 region,highlighted sequences represent the hinge region, regular sequencesrepresent the CH2 region and underlined sequences represent the CH3region

SEQ ID No: 8: The amino acid sequence of the IgG2 constant region(accession number P01859). Sequences in italics represent the CH1region, highlighted sequences represent the hinge region, regularsequences represent the CH2 region and underlined sequences representthe CH3 region

SEQ ID No: 9: The amino acid sequence of the IgG3 constant region(accession number A23511). Sequences in italics represent the CH1region, highlighted sequences represent the hinge region, regularsequences represent the CH2 region and underlined sequences representthe CH3 region

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “antibody” as referred to herein includes whole antibodymolecules, antigen binding fragments, monovalent antibodies, and singlechains thereof. Antibody molecules belong to a family of plasma proteinscalled immunoglobulins, whose basic building block, the immunoglobulinfold or domain, is used in various forms in many molecules of the immunesystem and other biological recognition systems. Native antibodies andimmunoglobulins are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries between the heavy chains of different immunoglobulin isotypes.Each heavy and light chain may also have regularly spaced intrachaindisulfide bridges. Each light chain is comprised of a light chainvariable region (abbreviated herein as VL) and a light chain constantregion (abbreviated herein as CL). Each heavy chain is comprised of aheavy chain variable region (VH) and a heavy chain constant region (CH)consisting of three domains, CH1, CH2 and CH3, and the hinge region).The three CH domains and the hinge region have been indicated for IgG1,IgG2, IgG3 and IgG4 in SEQ ID NO: 7, 8, 9 and 2, respectively (seebelow) The constant domain of the light chain is aligned with the firstconstant domain (CH1) of the heavy chain, and the light chain variabledomain is aligned with the variable domain of the heavy chain formingwhat is known as the “Fab fragment”. CH1 and CH2 of the heavy chain areseparated form each other by the so-called hinge region, which allowsthe Fab “arms” of the antibody molecule to swing to some degree. Thehinge region normally comprises one or more cysteine residues, which arecapable of forming disulphide bridges with the cysteine residues of thehinge region of the other heavy chain in the antibody molecule.

The variable regions of the heavy and light chains contain a bindingdomain that interacts with an antigen. The constant regions of theantibodies may mediate the binding of the immunoglobulin to host tissuesor factors, including various cells of the immune system (for instanceeffector cells) and the first component (C1q) of the classicalcomplement system

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five (5) major classes of immunoglobulins: IgA, IgD,IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), for instance IgG1, IgG2, IgG3 and IgG4; IgA1 andIgA2. The genes for the heavy chains constant domains that correspond tothe different classes of immunoglobulins are called alpha (α), delta(δ), epsilon (ε), gamma (γ) and mu (μ), respectively. Immunoglobulinsubclasses are encoded by different genes such as γ1, γ2, γ3 and γ4. Thegenes for the light chains of antibodies are assigned to one of twoclearly distinct types, called kappa (κ) and lambda (λ), based on theamino sequences of their constant domain. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. Distinct allotypes of immunoglobulins exist within thehuman population such as G1m(a), G1m(x), G1m(f) and G1m(z) for IgG1heavy chain and Km1, Km-1,2 and Km3 for the kappa light chain. Theseallotypes differ at distinct amino acids in their region encoding theconstant regions.

The term antibody also encompasses “derivatives” of antibodies, whereinone or more of the amino acid residues have been derivatised, forinstance by acylation or glycosylation, without significantly affectingor altering the binding characteristics of the antibody containing theamino acid sequences.

In addition, the term antibody covers variants, e.g. variants whereinthe in vivo half-life of the antibodies has been improved by modifyingthe salvage receptor epitope of the Ig constant domain or an Ig-likeconstant domain such that the molecule does not comprise an intact CH2domain or an intact Ig Fc region, cf. U.S. Pat. No. 6,121,022 and U.S.Pat. No. 6,194,551. The in vivo half-life may be furthermore increasedby making mutations in the Fc region, for instance by substitutingthreonine for leucine at the position corresponding to position 252 ofan intact antibody molecule, threonine for serine at the positioncorresponding to position 254 of an intact antibody molecule, orthreonine for phenylalanine at the position corresponding to position256 of an intact antibody molecule, cf. U.S. Pat. No. 6,277,375.

Furthermore, antibodies, and particularly Fab or other fragments, may bepegylated to increase the half-life. This can be carried out bypegylation reactions known in the art, as described, for example, inFocus on Growth Factors 3, 4-10 (1992), EP 154 316 and EP 401 384.

The term “antibody half-molecule” is used herein to mean an antibodymolecule as described above, but comprising no more than one light chainand no more than one heavy chain, and which exists in water solutions asa heterodimer of said single light and single heavy chain. Such antibodyis by nature monovalent as only one antigen-binding portion is present.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (for instance mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR1 or CDR2 sequences derived from thegermline of another mammalian species, such as a mouse, or the CDR3region derived from an antibody from another species, such as mouse,have been grafted onto human framework sequences. Human monoclonalantibodies directed may be generated using transgenic ortranschromosomal mice carrying parts of the human immune system ratherthan the mouse system. Such transgenic and transchromosomic mice includemice referred to herein as HuMAb mice and KM mice, respectively, and arecollectively referred to herein as “transgenic mice”.

The HuMAb mouse contains a human immunoglobulin gene miniloci thatencodes unrearranged human heavy (μ and γ) and K light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous p and K chain loci (Lonberg, N. et al., Nature368, 856-859 (1994)). Accordingly, the mice exhibit reduced expressionof mouse IgM or K and in response to immunization, the introduced humanheavy and light chain transgenes, undergo class switching and somaticmutation to generate high affinity human IgG,κ monoclonal antibodies(Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook ofExperimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar,D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. andLonberg, N. Ann. N.Y. Acad. Sci. 764 536-546 (1995)). The preparation ofHuMAb mice is described in detail in Taylor, L. et al., Nucleic AcidsResearch 20, 6287-6295 (1992), Chen, J. et al., International Immunology5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994),Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild,D. et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat.No. 5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126, U.S.Pat. No. 5,633,425, U.S. Pat. No. 5,789,650, U.S. Pat. No. 5,877,397,U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,814,318, U.S. Pat. No.5,874,299, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,545,807, WO98/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO01/09187.

The HCo7 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429).

The HCo12 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCo12 human heavy chain transgene (as described in Example 2 of WO01/14424).

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal stabilized IgG4 antibodiesaccording to well known techniques. Such transgenic non-human animals,non-human animals comprising an operable nucleic acid sequence codingfor expression of antibody used in the invention, non-human animalsstably transfected with one or more target-encoding nucleic acidsequences, and the like, are additional features of the presentinvention. The term “K_(D)” (M), as used herein, refers to thedissociation equilibrium constant of a particular antibody-antigeninteraction.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “human monoclonal antibody” refers to antibodiesdisplaying a single binding specificity which have variable and constantregions derived from human germline immunoglobulin sequences.

The term “monovalent antibody” means in the present context that anantibody molecule is capable of binding a single molecule of theantigen, and thus is not able of antigen crosslinking.

As used herein, “specific binding” refers to the binding of an antibody,or antigen-binding fragment thereof, to a predetermined antigen.Typically, the antibody binds with an affinity corresponding to a K_(D)of about 10⁻⁷ M or less, such as about 10⁻⁸ M or less, such as about10⁻⁹ M or less, about 10⁻¹⁰ M or less, or about 10⁻¹¹M or even less,when measured for instance using sulfon plasmon resonance on BIAcore oras apparent affinities based on IC₅₀ values in FACS or ELISA, and bindsto the predetermined antigen with an affinity corresponding to a K_(D)that is at least ten-fold lower, such as at least 100 fold lower, forinstance at least 1000 fold lower, such as at least 10,000 fold lower,for instance at least 100,000 fold lower than its affinity for bindingto a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The amount withwhich the affinity is lower is dependent on the K_(D) of the antigenbinding peptide, so that when the K_(D) of the antigen binding peptideis very low (that is, the antigen binding peptide is highly specific),then the amount with which the affinity for the antigen is lower thanthe affinity for a non-specific antigen may be at least 10,000 fold.

The terms “transgenic, non-human animal” refers to a non-human animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or transchromosomes (either integrated or non-integrated intothe animal's natural genomic DNA) and which is capable of expressinghuman antibodies. For example, a transgenic mouse can have a human lightchain transgene and either a human heavy chain transgene or human heavychain transchromosome, such that the mouse produces human antibodieswhen immunized with an antigen and/or cells expressing an antigen. Thehuman heavy chain transgene can be integrated into the chromosomal DNAof the mouse, as is the case for transgenic, for instance HuMAb mice,such as HCo7 or HCo12 mice, or the human heavy chain transgene can bemaintained extrachromosomally, as is the case for transchromosomal KMmice as described in WO 02/43478. Such transgenic and transchromosomalmice are capable of producing multiple classes and isotypes ofmonovalent antibodies to a given antigen (for instance IgM, IgG, IgAand/or IgE) by undergoing V-D-J recombination and isotype switching.

The term “acceptor site for N-linked glycosylation” refers to a site ona polypeptide which is susceptible of becoming glycosylated on an Asnresidue. The typical consensus site for this type of glycosylation isAsn-X-Ser/Thr, wherein X can be any amino acid, except for Pro.

As explained above, the characteristic IgG structure in which twoheavy-light chain heterodimers are linked is maintained by theinter-heavy chain disulphide bridges of the hinge region and thenon-covalent interactions of the CH3 domains.

It has been shown in WO2007059782 that removal of the hinge region inIgG4 results in the formation of monovalent antibodies in which thelinkage between the two heavy-light chain heterodimers is lost ordiminished. Consequently, changes in hinge region disulphide bridges ofother IgG subclasses alone or in combination with mutations in the CH3domain interactions may result in the formation of monovalent antibodiesfor these other subclasses as well. It is well within the capability ofthe skilled artisan to use the intimate knowledge of structure of Igsubclasses, and the knowledge provided in the present invention, toselect and to modify selected amino acids to prevent light chaininteractions.

In a first main aspect, the invention relates to a monovalent antibody,which comprises

(i) a variable region of a selected antigen specific antibody or anantigen binding part of the said region, and

(ii) a CH region of an immunoglobulin or a fragment thereof comprisingthe CH2 and CH3 regions, wherein the CH region or fragment thereof hasbeen modified such that the region corresponding to the hinge regionand, if the immunoglobulin is not an IgG4 subtype, other regions of theCH region, such as the CH3 region, do not comprise any amino acidresidues which are capable of forming disulfide bonds with an identicalCH region or other covalent or stable non-covalent inter-heavy chainbonds with an identical CH region in the presence of polyclonal humanIgG,

wherein the antibody is of the IgG4 type and the constant region of theheavy chain has been modified so that one or more of the following aminoacid substitutions have been made relative the sequence set forth in SEQID NO: 4: Tyr (Y) in position 217 has been replaced by Arg (R), Leu (L)in position 219 has been replaced by Asn (N) or Gln (Q), Glu (E) inposition 225 has been replaced by Thr (T), Val (V) or Ile (I), Ser (S)in position 232 has been replaced by Arg (R) or Lys (K), Thr (T) inposition 234 has been replaced by Arg (R), Lys (K) or Asn (N), Leu (L)in position 236 has been replaced by Ser (S) or Thr (T), Lys (K) inposition 238 has been replaced by Arg (R), Asp (D) in position 267 hasbeen replaced by Thr (T) or Ser (S), Phe (F) in position 273 has beenreplaced by Arg (R), Gln (Q), Lys (K) or Tyr (Y), Tyr (Y) in position275 has been replaced by Gln (Q), Lys (K) or Phe (F), Arg (R) inposition 277 has been replaced by Glu (E), Thr (T) in position 279 hasbeen replaced by Asp (D), Val (V) and Asn (N),

or the antibody is of another IgG type and the constant region of theheavy chain has been modified so that one or more of the same amino-acidsubstitutions have been made at the positions that correspond to thebefore-mentioned positions for IgG4. See e.g. SEQ ID NO: 7, 8 and 9 forthe corresponding positions in other isotypes.

In one embodiment, the monovalent antibody comprises

(i) a variable region of a selected antigen specific antibody or anantigen binding part of the said region, and

(ii) a CH region of an immunoglobulin or a fragment thereof comprisingthe CH2 and CH3 regions, wherein the CH region or fragment thereof hasbeen modified such that the region corresponding to the hinge regionand, if the immunoglobulin is not an IgG4 subtype, other regions of theCH region, such as the CH3 region, do not comprise any amino acidresidues which are capable of forming disulfide bonds with an identicalCH region or other covalent or stable non-covalent inter-heavy chainbonds with an identical CH region in the presence of polyclonal humanIgG,

wherein the antibody is of the IgG4 type and the constant region of theheavy chain has been modified so that one or more of the following aminoacid substitutions have been made relative the sequence set forth in SEQID NO: 4: Glu (E) in position 225 has been replaced by Val (V), Ser (S)in position 232 has been replaced by Arg (R), Leu (L) in position 236has been replaced by Ser (S) or Thr (T), Asp (D) in position 267 hasbeen replaced by Thr (T) or Ser (S), Phe (F) in position 273 has beenreplaced by Arg (R), Gln (Q) or Tyr (Y), Tyr (Y) in position 275 hasbeen replaced by Gln (Q) or Lys (K).

In another embodiment, the antibody is of the IgG4 type and the constantregion of the heavy chain has been modified so that one or more of thefollowing combinations of amino acid substitutions have been maderelative the sequence set forth in SEQ ID NO: 4:

Asp (D) in position 267 has been replaced by Ser (S) and Tyr (Y) inposition 275 has been replaced by Gln (Q) or Lys (K), Arg (R),

Asp (D) in position 267 has been replaced by Thr (T) and Tyr (Y) inposition 275 has been replaced by Gln (Q) or Lys (K), Arg (R),

or the antibody is of another IgG type and the constant region of theheavy chain has been modified so that the same combinations ofamino-acid substitutions have been made at the positions that correspondto the before-mentioned positions for IgG4.

Typically, the variable region and the C_(H) region of the monovalentantibody are connected to each other via peptide bonds and are producedfrom a single open reading frame. Without being bound to any theory, itis believed that the monovalent antibodies according to the inventionare capable of binding to the FcRn. Such binding may be determined byuse of methods for determining binding as it is known in the art, forinstance by use of ELISA assays. The binding of a monovalent antibody ofthe invention to FcRn may for instance be compared to the binding of aF(ab′)₂ fragment, which F(ab′)₂ fragment has a VH region and a VLregion, which are identical to the VH region and the VL region of themonovalent antibody of the invention, to FcRn in the same assay. In oneembodiment, the binding of a monovalent antibody of the invention toFcRn is more than 10 times stronger than the binding of the F(ab′)₂fragment to FcRn.

In one embodiment, the antibody (further) comprises a CH1 region.

In another embodiment, the monovalent antibody consists of said variableregion and said CH region.

In another embodiment, the variable region is a VH region. In a furtherembodiment, the variable region is a VL region. In an even furtherembodiment, the antibody does not comprise a CL region.

In an important embodiment, the monovalent antibody of the inventioncomprises a heavy chain and a light chain, wherein the heavy chaincomprises

-   -   (i) a VH region of a selected antigen specific antibody or an        antigen binding part of the said region, and    -   (ii) a CH region as defined above,        and the light chain comprises    -   (i) a VL region of a selected antigen specific antibody or an        antigen binding part of the said region, and    -   (ii) a CL region which, in case of an IgG1 subtype, has been        modified such that the CL region does not contain any amino        acids, which are capable of forming disulfide bonds with an        identical CL region or other covalent bonds with an identical CL        region in the presence of polyclonal human IgG.

Typically, the light chain and the heavy chain of the monovalentantibody defined above are connected to each other via one or moredisulfide bonds. It is evident that for such disulphide bonds, neitherof the binding partners in the disulphide bond is present in the regioncorresponding to the hinge region. In one embodiment however the lightchain and the heavy chain of the monovalent antibody are connected toeach other via one or more amide bonds.

Furthermore, typically, the VL region and the CL region of the lightchain are connected to each other via peptide bonds and produced from asingle open reading frame.

In one embodiment, the VH and VL region of an antibody molecule of theinvention are derived from the same antigen specific antibody.

According to the invention, the sequence of the CL region of the lightchain of the antibody molecule may be derived from the sequence of CLregion of an immunoglobulin. In one embodiment, the CL region is theconstant region of the kappa light chain of human IgG. In oneembodiment, the CL region comprises the amino acid sequence of SEQ IDNo: 2. In one embodiment, the CL region is the constant region of thelambda light chain of human IgG. In one embodiment, the CL regioncomprises the amino acid sequence of SEQ ID No: 4.

In one embodiment, the monovalent antibody of the invention is an IgG1,IgG2, IgG3, IgG4, IgA or IgD antibody, such as an IgG1, IgG2 or IgG4antibody. In a further embodiment, the monovalent antibody is a humanantibody.

A monovalent antibody of the present invention may also be a variant ofany of the above isotypes. For example, a variant IgG4 antibody may bean antibody that differs from a IgG4 antibody by one or more suitableamino acid residue alterations, that is substitutions, deletions,insertions, or terminal sequence additions, for instance in the constantdomain, and/or the variable regions (or any one or more CDRs thereof) ina single variant antibody. Typically, amino acid sequence alterations,desirably do not substantially change the structural characteristics ofthe parent sequence (e.g., a replacement amino acid should not tend todisrupt secondary structure that characterizes the function of theparent sequence), but which may be associated with advantageousproperties, such as changing the functional or pharmacokineticproperties of the antibodies, for example increasing the half-life,altering the immunogenicity, providing a site for covalent ornon-covalent binding to another molecule, reducing susceptibility toproteolysis or reducing susceptibility to oxidation. Examples ofvariants include variants which have a modification of the CH3 region,such as a substitution or deletion at any one or more of the positions225, 234, 236, 238, 273 or 275 of SEQ ID NO: 4 or the correspondingresidues in non-IgG4 isotypes. Modifications at these positions may e.g.further reduce intermolecular interactions between hinge-modifiedantibodies of the invention. Other examples include variants which havea modification of the constant region, such as a substitution ordeletion, at any one or more of the positions 118, 120, 122, 124, 175,248, 296, 302 of SEQ ID NO: 4 or the corresponding residues in non-IgG4isotypes. Modifications at these positions may e.g. increase thehalf-life of hinge-modified antibodies of the invention.

In one embodiment, the monovalent antibody of the invention comprisesthe CH3 region as set as set forth in SEQ ID NO: 7, but wherein the CH3region has been modified so that one or more of the following amino acidsubstitutions have been made: Arg (R) in position 238 has been replacedby Gln (Q); Asp (D) in position 239 has been replaced by Glu (E); Thr(T) in position 249 has been replaced by Ala (A); Leu (L) in position251 has been replaced by Ala (A); Leu (L) in position 251 has beenreplaced by Val (V); Phe (F) in position 288 has been replaced by Ala(A); Phe (F) in position 288 has been replaced by Leu (L); Tyr (Y) inposition 290 has been replaced by Ala (A); Lys (K) in position 292 hasbeen replaced by Arg (R); Lys (K) in position 292 has been replaced byAla (A); Gln (Q) in position 302 has been replaced by Glu (E); and Pro(P) in position 328 has been replaced by Leu (L).

In a further embodiment hereof, one or more of the following amino acidsubstitutions have been made: Arg (R) in position 238 has been replacedby Gln (Q); Asp (D) in position 239 has been replaced by Glu (E); Lys(K) in position 292 has been replaced by Arg (R); Gln (Q) in position302 has been replaced by Glu (E); and Pro (P) in position 328 has beenreplaced by Leu (L). In an even further embodiment:

(i) Arg (R) in position 238 has been replaced by Gln (Q),(ii) Arg (R) in position 238 has been replaced by Gln (Q), and Pro (P)in position 328 has been replaced by Leu (L), or(iii) all five substitutions as defined above have been made.

In another further embodiment hereof, the monovalent antibody furthercomprises the CH1 and/or CH2 regions as set forth in SEQ ID NO: 7, withthe proviso that the CH2 region has been modified so that it does notcomprise any acceptor sites for N-linked glycosylation.

In one embodiment, the monovalent antibody of the invention comprisesthe kappa CL region having the amino acid sequence as set forth in SEQID NO: 6, but wherein the sequence has been modified so that theterminal cysteine residue in position 106 has been replaced with anotheramino acid residue or has been deleted.

In another embodiment, the monovalent antibody of the inventioncomprises the lambda CL region having the amino acid sequence as setforth in SEQ ID NO: 5, but wherein the sequence has been modified sothat the cysteine residue in position 104 has been replaced with anotheramino acid residue or has been deleted.

In a further embodiment, the monovalent antibody of the inventioncomprises the CH1 region as set forth in SEQ ID NO: 7, but wherein theCH1 region has been modified so that Ser (S) in position 14 has beenreplaced by a cysteine residue.

In a different embodiment, the monovalent antibody of the inventioncomprises the CH3 region as set forth in SEQ ID NO: 8, but wherein theCH3 region has been modified so that one or more of the of the followingamino acid substitutions have been made: Arg (R) in position 234 hasbeen replaced by Gln (Q); Thr (T) in position 245 has been replaced byAla (A); Leu (L) in position 247 has been replaced by Ala (A); Leu (L)in position 247 has been replaced by Val (V); Met (M) in position 276has been replaced by Val (V); Phe (F) in position 284 has been replacedby Ala (A); Phe (F) in position 284 has been replaced by Leu (L); Tyr(Y) in position 286 has been replaced by Ala (A); Lys (K) in position288 has been replaced by Arg (R); Lys (K) in position 288 has beenreplaced by Ala (A); Gln (Q) in position 298 has been replaced by Glu(E); and Pro (P) in position 324 has been replaced by Leu (L).

In a further embodiment hereof, one or more of the of the followingamino acid substitutions have been made: Arg (R) in position 234 hasbeen replaced by Gln (Q); Met (M) in position 276 has been replaced byVal (V); Lys (K) in position 288 has been replaced by Arg (R); Gln (Q)in position 298 has been replaced by Glu (E); and Pro (P) in position324 has been replaced by Leu (L). In an even further embodiment:

(i) Arg (R) in position 234 has been replaced by Gln (Q);(ii) Arg (R) in position 234 has been replaced by Gln (Q); and Pro (P)in position 324 has been replaced by Leu (L); or(iii) all five substitutions as defined above have been made.

In another further embodiment hereof, the monovalent antibody furthercomprises the CH1 and/or CH2 regions as set forth in SEQ ID NO: 8, withthe proviso that the CH2 region has been modified so that it does notcomprise any acceptor sites for N-linked glycosylation.

In a further different embodiment, the monovalent antibody of theinvention comprises the CH3 region as set forth in SEQ ID NO: 9, butwherein the CH3 region has been modified so that one or more of thefollowing amino acid substitutions have been made: Arg (R) in position285 has been replaced by Gln (Q); Thr (T) in position 296 has beenreplaced by Ala (A); Leu (L) in position 298 has been replaced by Ala(A); Leu (L) in position 298 has been replaced by Val (V); Ser (S) inposition 314 has been replaced by Asn (N); Asn (N) in position 322 hasbeen replaced by Lys (K); Met (M) in position 327 has been replaced byVal (V); Phe (F) in position 335 has been replaced by Ala (A); Phe (F)in position 335 has been replaced by Leu (L); Tyr (Y) in position 337has been replaced by Ala (A); Lys (K) in position 339 has been replacedby Arg (R); Lys (K) in position 339 has been replaced by Ala (A); Gln(Q) in position 349 has been replaced by Glu (E); Ile (I) in position352 has been replaced by Val (V); Arg (R) in position 365 has beenreplaced by His (H); Phe (F) in position 366 has been replaced by Tyr(Y); and Pro (P) in position 375 has been replaced by Leu (L), with theproviso that the CH3 region has been modified so that it does notcomprise any acceptor sites for N-linked glycosylation.

In a further embodiment hereof, one or more of the of the followingamino acid substitutions have been made: Arg (R) in position 285 hasbeen replaced by Gln (Q); Ser (S) in position 314 has been replaced byAsn (N); Asn (N) in position 322 has been replaced by Lys (K); Met (M)in position 327 has been replaced by Val (V); Lys (K) in position 339has been replaced by Arg (R); Gln (Q) in position 349 has been replacedby Glu (E); Ile (I) in position 352 has been replaced by Val (V); Arg(R) in position 365 has been replaced by His (H); Phe (F) in position366 has been replaced by Tyr (Y); and Pro (P) in position 375 has beenreplaced by Leu (L). In an even further embodiment:

(i) Arg (R) in position 285 has been replaced by Gln (Q),(ii) Arg (R) in position 285 has been replaced by Gln (Q); and Pro (P)in position 375 has been replaced by Leu (L), or(iii) all ten substitutions as defined above have been made.

In another further embodiment hereof, the monovalent antibody furthercomprises the CH1 and/or CH2 regions as set forth in SEQ ID NO: 9, withthe proviso that the CH2 region has been modified so that it does notcomprise any acceptor sites for N-linked glycosylation.

In further embodiments, the monovalent antibody according to theinvention has been further modified e.g. in the CH2 and/or CH3 region,for example, to reduce the ability of the monovalent antibody todimerize or to improve the pharmacokinetic profile, e.g. via improvingthe binding to FcRn.

Examples of such modifications include the following substitutions(reference is here made to IgG4 residues given in SEQ ID NO:4, but thesame substitutions may be made in corresppnding residues in otherisotypes, such as IgG1. These corresponding residues may be found bysimply alignment of the sequence): in the CH3 region: T234A, L236A,L236V, F273A, F273L, Y275A, E225A, D267A, L236E, L236G, F273D, F273T,Y275E, and in the CH2 region: T118Q, M296L, M120Y, S122T, T124E, N₃₀₂A,T175A, E248A, N302A. Two or more of the above mentioned substitutionsmade combined to obtain the combined effects.

Thus, in one embodiment, the monovalent antibody comprises the CH3region as set forth in SEQ ID NO: 4.

However, in another embodiment, the monovalent antibody comprises theCH3 region as set forth in SEQ ID NO: 4, but:

Glu (E) in position 225 has been replaced by Ala (A), and/or

Thr (T) in position 234 has been replaced by Ala (A), and/or

Leu (L) in position 236 has been replaced by Ala (A), Val (V), Glu (E)or Gly (G), and/or

Asp (D) in position 267 has been replaced by Ala (A), and/or

Phe (F) in position 273 has been replaced by Ala (A) or Leu (L).

Tyr (Y) in position 275 has been replaced by Ala (A).

In another embodiment, the monovalent antibody comprises the CH3 regionas set forth in SEQ ID NO: 4, but:

Glu (E) in position 225 has been replaced by Ala (A), and/or

Thr (T) in position 234 has been replaced by Ala (A), and/or

Leu (L) in position 236 has been replaced by Ala (A), Val (V), Glu (E)or Gly (G), and/or

Asp (D) in position 267 has been replaced by Ala (A), and/or

Phe (F) in position 273 has been replaced by Asp (D) and Tyr (Y) inposition 275 has been replaced by Glu (E).

In another embodiment, the monovalent antibody comprises the CH3 regionas set forth in SEQ ID NO: 4, but:

Glu (E) in position 225 has been replaced by Ala (A), and/or

Thr (T) in position 234 has been replaced by Ala (A), and/or

Leu (L) in position 236 has been replaced by Ala (A), Val (V), Glu (E)or Gly (G), and/or

Asp (D) in position 267 has been replaced by Ala (A), and/or

Phe (F) in position 273 has been replaced by Thr (T) and Tyr (Y) inposition 275 has been replaced by Glu (E).

In one embodiment, the monovalent antibody comprises the CH2 region asset forth in SEQ ID NO: 4, but wherein Thr (T) in position 118 has beenreplaced by Gln (Q) and/or Met (M) in position 296 has been replaced byLeu (L).

In another embodiment, the monovalent antibody comprises the CH2 regionas set forth in SEQ ID NO: 4, but wherein one, two or all three of thefollowing substitutions have been made: Met (M) in position 120 has beenreplaced by Tyr (Y); Ser (S) in position 122 has been replaced by Thr(T); and Thr (T) in position 124 has been replaced by Glu (E).

In another embodiment, the monovalent antibody comprises the CH2 regionas set forth in SEQ ID NO: 4, but wherein Asn (N) in position 302 hasbeen replaced by Ala (A).

In a yet other embodiment, the monovalent antibody comprises the CH2region as set forth in SEQ ID NO: 4, but wherein Asn (N) in position 302has been replaced by Ala (A) and Thr (T) in position 175 has beenreplaced by Ala (A) and Glu (E) in position 248 has been replaced by Ala(A).

In an even further different embodiment, the antibody of the inventioncomprises the CH3 region as set forth in SEQ ID NO: 4, and wherein theCH3 region has been modified so that one or more of the following aminoacid substitutions have been made: Thr (T) in position 234 has beenreplaced by Ala (A); Leu (L) in position 236 has been replaced by Ala(A); Leu (L) in position 236 has been replaced by Val (V); Phe (F) inposition 273 has been replaced by Ala (A); Phe (F) in position 273 hasbeen replaced by Leu (L); Tyr (Y) in position 275 has been replaced byAla (A); Arg (R) in position 277 has been replaced by Ala (A).

Preferred substitutions include: replacement of Leu (L) in position 236by Val (V), replacement of Phe (F) in position 273 by Ala (A) andreplacement of Tyr (Y) in position 275 by Ala (A).

In one embodiment of the invention, the monovalent antibody does notbind to the synthetic antigen (Tyr, Glu)-Ala-Lys.

The hinge region is a region of an antibody situated between the CH1 andCH2 regions of the constant domain of the heavy chain. The extent of thehinge region is determined by the separate exon, which encodes the hingeregion. The hinge region is normally involved in participating inensuring the correct assembly of the four peptide chains of an antibodyinto the traditional tetrameric form via the formation of disulphidebonds, or bridges, between one or more cysteine residues in the hingeregion of one of the heavy chains and one or more cysteine residues inthe hinge region of the other heavy chain. A modification of the hingeregion so that none of the amino acid residues in the hinge region arecapable of participating in the formation of disulphide bonds may thusfor instance comprise the deletion and/or substitution of the cysteineresidues present in the unmodified hinge region. A region correspondingto the hinge region should for the purpose of this specification beconstrued to mean the region between region CH1 and CH2 of a heavy chainof an antibody. In the context of the present invention, such a regionmay also comprise no amino acid residues at all, corresponding to adeletion of the hinge region, resulting in the CH1 and CH2 regions beingconnected to each other without any intervening amino acid residues.Such a region may also comprise only one or a few amino acid residues,which residues need not be the amino acid residues present in the N- orC-terminal of the original hinge region.

Accordingly, in one embodiment of the antibody of the invention, the CHregion has been modified such that the region corresponding to the hingeregion of the CH region does not comprise any cysteine residues. Inanother embodiment, the CH region has been modified such that at leastall cysteine residues have been deleted and/or substituted with otheramino acid residues. In a further embodiment, the CH region has beenmodified such that the cysteine residues of the hinge region have beensubstituted with amino acid residues that have an uncharged polar sidechain or a nonpolar side chain. Preferably, the amino acids withuncharged polar side chains are independently selected from asparagine,glutamine, serine, threonine, tyrosine, and tryptophan, and the aminoacid with the nonpolar side chain are independently selected fromalanine, valine, leucine, isoleucine, proline, phenylalanine, andmethionine.

In an even further embodiment, the monovalent antibody is a human IgG4,wherein the amino acids corresponding to amino acids 106 and 109 of theCH sequence of SEQ ID No: 2 have been deleted.

In a yet further embodiment, the monovalent antibody is a human IgG4,wherein one of the amino acid residues corresponding to amino acidresidues 106 and 109 of the sequence of SEQ ID No: 2 has beensubstituted with an amino acid residue different from cysteine, and theother of the amino acid residues corresponding to amino acid residues106 and 109 of the sequence of SEQ ID No: 2 has been deleted.

In a yet further embodiment, the amino acid residue corresponding toamino acid residue 106 has been substituted with an amino acid residuedifferent from cysteine, and the amino acid residue corresponding toamino acid residue 109 has been deleted.

In a yet further embodiment, the amino acid residue corresponding toamino acid residue 106 has been deleted, and the amino acid residuecorresponding to amino acid residue 109 has been substituted with anamino acid residue different from cysteine.

In a yet further embodiment, the monovalent antibody is a human IgG4,wherein at least the amino acid residues corresponding to amino acidresidues 106 to 109 of the CH sequence of SEQ ID No: 2 have beendeleted.

In a yet further embodiment, the monovalent antibody is a human IgG4,wherein at least the amino acid residues corresponding to amino acidresidues 99 to 110 of the sequence of SEQ ID No: 2 have been deleted.

In a yet further embodiment, the CH region comprises the amino acidsequence of SEQ ID No: 4.

In a yet even further embodiment, the monovalent antibody is a humanIgG4, wherein the CH region has been modified such that the entire hingeregion has been deleted.

In a further embodiment, the sequence of the antibody has been modifiedso that it does not comprise any acceptor sites for N-linkedglycosylation. In a further embodiment hereof, the NST acceptor site forN-linked glycosylation in the CH2 region has been modified to a sequenceselected from the group consisting of: GST, MST, CSE, DSE, DSP, ESP,GSP, HSE, NSE, PSP and SSE.

In one embodiment, the monovalent antibody of the invention ismonovalent in the presence of physiological concentrations of polyclonalhuman IgG.

The antibodies of the present invention has the advantage of having along half-life in vivo, leading to a longer therapeutic window, ascompared to e.g. a FAB fragment of the same antibody which has aconsiderably shorter half-life in vivo.

Further, due to the long half-life and small size, the monovalentantibodies of the invention will have a potential having a betterdistribution in vivo, in example by being able to penetrate solidtumors. This leads to a great use potential of the monovalent antibodiesof the invention, e.g. for treatment of cancer, since the antibodies ofthe invention could be used either to inhibit a target molecule, or as atarget specific delivery mechanism for other drugs that would treat thedisease.

Accordingly, in one embodiment, the monovalent antibody of the inventionhas a plasma concentration above 10 μg/ml for more than 7 days whenadministered in vivo at a dose of 4 mg per kg, as measured in anpharmacokinetic study in SCID mice (for instance as shown in theWO2007059782). The clearance rate of a monovalent antibody of theinvention may be measured by use of pharmacokinetic methods as it isknown in the art. The antibody may for instance be injectedintravenously (other routes such as i.p. or i.m. may also be used) in ahuman or animal after which blood samples are drawn by venipuncture atseveral time points, for instance 1 hour, 4 hours, 24 hours, 3 days, 7days, 14 days, 21 days and 28 days after initial injection). Theconcentration of antibody in the serum is determined by an appropriateassay such as ELISA. Pharmacokinetic analysis can performed as known inthe art and described in WO2007059782. Monovalent antibodies of theinvention may have a plasma residence time, which is as much as 100times longer than the plasma residence time of for instance Fabfragments which are frequently used as monovalent antibodies.

In one embodiment, a monovalent antibody of the invention has a plasmaclearance, which is more than 10 times slower than the plasma clearanceof a F(ab′)₂ fragment, which has a comparable molecular size. This maybe an indication of the capability of the antibodies of the invention tobind to FcRn. FcRn is a major histocompatibility complex class I-relatedreceptor and plays a role in the passive delivery of immunoglobulin(Ig)Gs from mother to young and in the regulation of serum IgG levels byprotecting IgG from intracellular degradation (Ghetie V et al., Annu RevImmunol. 18, 739-66 (2000)). In one embodiment, the F(ab′)₂ fragment isdirected at the same antigen as the monovalent antibody of theinvention. In one embodiment, the F(ab′)₂ fragment is directed at thesame epitope as the monovalent antibody of the invention. In oneembodiment, the VH region and the VL region of the F(ab′)₂ fragment areidentical to the VH region and the VL region of the monovalent antibodyof the invention.

In one embodiment, a monovalent antibody of the invention has ahalf-life of at least 5 days when administered in vivo. The half-life ofa monovalent antibody of the invention may be measured by any methodknown in the art, for instance as described above.

In one embodiment, a monovalent antibody of the invention has ahalf-life of at least 5 days and up to 14 days, when administered invivo.

In one embodiment, the monovalent antibody of the invention has ahalf-life of at least 5 days and up to 21 days, when administered invivo.

In an even further embodiment, the monovalent antibody has a serumhalf-life of at least 5 days, such as of at least 14 days, for exampleof from 5 and up to 21 days when administered in vivo to a human beingor a SCID mouse.

In one embodiment, the monovalent antibody of the invention binds to atumor antigen with a dissociation constant (k_(d)) of 10⁻⁷ M or less,such as 10⁻⁸ M or less.

In another embodiment, the monovalent antibody of the invention binds toa cell surface receptor with a dissociation constant (k_(d)) of 10⁻⁷ Mor less, such as 10⁻⁸ M or less, which cell surface receptor isactivated upon receptor dimerization.

In a further embodiment, the monovalent antibody binds to a target witha dissociation constant (k_(d)) of 10⁻⁷ M or less, such as 10⁻⁸ M orless, which target is selected from: erythropoietin, beta-amyloid,thrombopoietin, interferon-alpha (2a and 2b), -beta (1b), -gamma, TNFR I(CD120a), TNFR II (CD120b), IL-1R type 1 (CD121a), IL-1R type 2(CD121b), IL-2, IL2R (CD25), IL-2R-beta (CD123), IL-3, IL-4, IL-3R(CD123), IL-4R (CD124), IL-5R (CD125), IL-6R-alpha (CD126), -beta(CD130), IL-10, IL-11, IL-15BP, IL-15R, IL-20, IL-21, TCR variablechain, RANK, RANK-L, CTLA4, CXCR4R, CCR5R, TGF-betas, -beta2, -beta3,G-CSF, GM-CSF, MIF-R (CD74), M-CSF-R (CD115), GM-CSFR (CD116), solubleFcRI, sFcRII, sFcRIII, FcRn, Factor VII, Factor VIII, Factor IX, VEGF,VEGFxxxb, anti-psychotic drugs, anti-depressant drugs, anti-Parkinsondrugs, anti-seizure agents, neuromuscular blocking drugs, anti-epilepticdrugs, adrenocorticosteroids, insulin, proteins or enzymes involved inregulation of insulin, incretins (GIP and GLP-1) or drugs mimickingincretin action such as Exenatide and sitagliptin, thyroid hormones,growth hormone, ACTH, oestrogen, testosterone, anti-diuretic hormone,diuretics, blood products such as heparin and EPO, beta-blocking agents,cytotoxic agents, anti-viral drugs, anti-bacterial agents, anti-fungalagents, anti-parasitic drugs, anti-coagulation drugs, anti-inflammatorydrugs, anti-asthma drugs, anti-COPD drugs, Viagra, opiates, morphine,vitamins (such as vitamin C for conservation), hormones involved inpregnancy such as LH and FSH, hormones involved in sex changes,anti-conceptives and antibodies.

In one embodiment, a monovalent antibody of the invention specificallybinds a cell surface receptor that is activated upon receptordimerization. Monovalent antibodies, such as the monovalent antibodiesof the invention, may often be useful in the treatment of diseases ordisorders, where receptor activation is undesirable, since the antibodymolecules of the inventions due to their monovalent nature are unable toinduce such dimerization and thereby such activation. Without beinglimited to specific receptors, examples of such receptors could beerb-B1, erb-B2, erb-B3, erb-B4 and members of the ephrins and ephrinreceptors such as ephrin-A1 through A6, ephA1 through A8, ephrin B1through B3 and eph-B1 through eph-B6.

In one embodiment, a monovalent antibody of the invention, when bound toa target molecule, inhibits target molecule multimerization (such asdimerization). Again, monovalent antibodies, such as the monovalentantibodies of the invention, may often be useful in the treatment ofdiseases or disorders, where multimerization of the target antigen isundesirable, since the antibody molecules of the inventions due to theirmonovalent nature are unable to induce such multimerization. In the caseof soluble antigens, multimerization may form undesirable immunecomplexes. Without being limited to specific targets, examples of suchtargets could be Toll-like receptors such as TLR-3 and TLR-9, orangiopoietin-1, or angiopoietin-2, or TNF receptor family members suchas CD30, CD40 and CD95.

In one embodiment, a monovalent antibody of the invention is aninhibitor of TNF-alpha. In one embodiment of the invention, themonovalent antibody of the invention is a monovalent form of adalimumab,etanercept, or infliximab.

In a further embodiment, the monovalent antibody binds to a target witha dissociation constant (k_(d)) of 10⁻⁷ M or less, such as 10⁻⁸ M orless, which target is selected from VEGF, c-Met, CD20, CD38, IL-8, CD25,CD74, FcalphaRI, FcepsilonRl, acetyl choline receptor, f as, fasL,TRAIL, hepatitis virus, hepatitis C virus, envelope E2 of hepatitis Cvirus, tissue factor, a complex of tissue factor and Factor VII, EGFr,CD4, and CD28.

In one embodiment, an anti-VEGF monovalent antibody is used fortreatment of AMD (acute macular degeneration), and other diseases.

In one embodiment, the anti-VEGF monovalent antibody used is amonovalent form of bevacizumab (Avastin).

In an even further embodiment, the monovalent antibody is a human IgG4antibody and which binds to c-Met with a dissociation constant (k_(d))of 10⁻⁷ M or less, such as 10⁻⁸ M or less.'

In one embodiment, a monovalent antibody of the invention is incapableof effector binding. The expression “incapable of effector binding” or“inability of effector binding” in the present context means that amonovalent antibody of the invention is incapable of binding to the C1qcomponent of the first component of complement (C1) and therefore isunable of activating the classical pathway of complement mediatedcytotoxicity. In addition, the monovalent antibodies of the inventionare unable to interact with Fc receptors and may therefore be unable totrigger Fc receptor-mediated effector functions such as phagocytosis,cell activation, induction of cytokine release

In one embodiment, a monovalent antibody of the invention is produced byuse of recombinant DNA technologies. Antibodies may be produced usingrecombinant eukaryotic host cells, such as Chinese hamster ovary (CHO)cells, NS/0 cells, HEK293 cells, insect cells, plant cells, or fungi,including yeast cells. Both stable as well as transient systems may beused for this purpose. Transfection may be done using plasmid expressionvectors by a number of established methods, such as electroporation,lipofection or nucleofection. Alternatively, infection may be used toexpress proteins encoded by recombinant viruses such as adeno, vacciniaor baculoviruses. Another method may be to use transgenic animals forproduction of antibodies.

Thus, in a further main aspect, the invention relates to a nucleic acidconstruct encoding the monovalent antibody of the invention as describedherein. In one embodiment, said nucleic acid construct is an expressionvector.

Furthermore, the invention relates to a method of preparing a monovalentantibody according to the invention comprising culturing a host cellcomprising a nucleic acid construct according to invention, so that themonovalent antibody is produced, and recovering the said monovalentantibody from the cell culture.

A DNA sequence encoding the antibody may be prepared synthetically byestablished standard methods. The DNA sequence may then be inserted intoa recombinant expression vector, which may be any vector, which mayconveniently be subjected to recombinant DNA procedures. The choice ofvector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e. a vector that exists as an extrachromosomal entity, the replicationof which is independent of chromosomal replication, for instance aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated. Inthe vector, a DNA sequence encoding the antibody should be operablyconnected to a suitable promoter sequence. The coding DNA sequence mayalso be operably connected to a suitable terminator and the vector mayfurther comprise elements such as polyadenylation signals (for instancefrom SV40 or the adenovirus 5 Elb region), transcriptional enhancersequences (for instance the SV40 enhancer) and translational enhancersequences (for instance the ones encoding adenovirus VA RNAs). Othersuch signals and enhancers are known in the art.

To obtain recombinant monovalent antibodies of the invention, the DNAsequences encoding different parts of the polypeptide chain(s) of theantibody may be individually expressed in a host cell, or may be fused,giving a DNA construct encoding the fusion polypeptide, such as apolypeptide comprising both light and heavy chains, inserted into arecombinant expression vector, and expressed in host cells.

Thus, in a further aspect, the invention relates to a host cellcomprising a nucleic acid according to the invention.

The invention also relate to a non-human transgenic animal comprising anucleic acid construct according to the invention.

The host cell into which the expression vector may be introduced, may beany cell which is capable of expression of full-length proteins, and mayfor instance be a prokaryotic or eukaryotic cell, such as yeast, insector mammalian cells. Examples of suitable mammalian cell lines are theHEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC CRL-1632, ATCCCCL-10), NS/0 (ECACC 85110503) or CHO (ATCC CCL-61) cell lines. Othersuitable cell lines are known in the art. In one embodiment, theexpression system is a mammalian expression system, such as a mammaliancell expression system comprising various clonal variations of HEK293cells.

Methods of transfecting mammalian cells and expressing DNA sequencesintroduced in the cells are well known in the art. To obtain amonovalent antibody of the invention, host cells of the expressionsystem may in one embodiment to be cotransfected with two expressionvectors simultaneously, wherein first of said two expression vectorscomprises a DNA sequence encoding the heavy chain of the antibody, andsecond of said two expression vectors comprises a DNA sequence encodingthe light chain of the antibody. The two sequences may also be presenton the same expression vector, or they may be fused giving a DNAconstruct encoding the fusion polypeptide, such as a polypeptidecomprising both light and heavy chains.

The recombinantly produced monovalent antibody may then be recoveredfrom the culture medium by conventional procedures including separatingthe host cells from the medium by centrifugation or filtration,precipitating the proteinaceous components of the supernatant orfiltrate by means of a salt, for instance ammonium sulphate,purification by a variety of chromatographic procedures, for instanceHPLC, ion exchange chromatography, affinity chromatography, Protein Achromatography, Protein G chromatography, or the like.

The present invention also relates to a method of preparing a monovalentantibody of the invention, wherein said method comprises the steps of:

-   -   (a) culturing a host cell comprising a nucleic acid encoding        said monovalent antibody; and    -   (b) recovering the monovalent antibody from the host cell        culture.

In one embodiment, said host cell is a prokaryotic host cell. In oneembodiment, the host cell is an E. coli cell. In one embodiment, the E.coli cells are of a strain deficient in endogenous protease activities.

In one embodiment, said host cell is a eukaryotic cell. In oneembodiment, the host cell is a HEK-293F cell. In another embodiment, thehost cell is a CHO cell.

In one embodiment, the monovalent antibody is recovered from culturemedium. In another embodiment, the monovalent antibody is recovered fromcell lysate.

In a further main aspect, the invention relates to a pharmaceuticalcomposition comprising the monovalent antibody according to theinvention. In one embodiment, the composition further comprises one ormore further therapeutic agents described herein.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 19^(th)Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995. As usedherein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible.

The pharmaceutical composition may be administered by any suitable routeand mode. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults.

In one embodiment, the pharmaceutical composition is suitable forparenteral administration. The phrase “parenteral administration” meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion. In one embodiment thepharmaceutical composition is administered by intravenous orsubcutaneous injection or infusion.

Regardless of the route of administration selected, the monovalentantibodies of the present invention, which may be used in the form of apharmaceutically acceptable salt or in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Dosage regimens are adjusted to provide the optimum desired response(for instance a therapeutic response). For example, a single bolus maybe administered, several divided doses may be administered over time orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofmonovalent antibody calculated to produce the desired therapeutic effectin association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the invention are dictated byand directly dependent on (a) the unique characteristics of themonovalent antibody and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch a monovalent antibody for the treatment of sensitivity inindividuals.

Actual dosage levels of the monovalent antibodies in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of theparticular monovalent antibodies of the present invention employed, theroute of administration, the time of administration, the rate ofexcretion of the particular monovalent antibody being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitable doseof a pharmaceutical composition of the invention will be that amount ofthe monovalent antibody which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. As another example, the physician orveterinarian may start with a high loading dose followed by repeatedadministration of lower doses to rapidly build up a therapeuticallyeffective dose and maintain it over longer periods of time.

A pharmaceutical composition of the invention may contain one or acombination of different monovalent antibodies of the invention. Thus,in a further embodiment, the pharmaceutical compositions include acombination of multiple (for instance two or more) monovalent antibodiesof the invention which act by different mechanisms. The monovalentantibodies may also be thus combined with divalent antibodies.

The monovalent antibody of the present invention have numerous in vitroand in vivo diagnostic and therapeutic utilities involving the diagnosisand treatment of disorders involving cells expressing the antigen whichthe antibody can recognize and bind to. In certain pathologicalconditions, it is necessary and/or desirable to utilize monovalentantibodies. Also, in some instances, it is preferred that a therapeuticantibody effects its therapeutic action without involving immunesystem-mediated activities, such as the effector functions, ADCC,phagocytosis and CDC. In such situations, it is desirable to generateforms of antibodies in which such activities are substantially reducedor eliminated. It is also advantageous if the antibody is of a form thatcan be made efficiently and with high yield. The present inventionprovides such antibodies, which may be used for a variety of purposes,for example as therapeutics, prophylactics and diagnostics.

In one embodiment, a monovalent antibody of the invention is directed toCD74 and inhibits MIF-induced signaling, but lacks Fc-mediated effectorfunctions.

In one embodiment, a monovalent antibody of the invention may preventbinding of a virus or other pathogen to its receptor, such as inhibitionof HIV binding to CD4 or coreceptor such as CCR5 or CXCR4.

The scientific literature is abundant with examples of targets, wherethe binding of antibodies against said target, or specific epitopes ofsaid target, is shown to have, or is expected to have, a therapeuticeffect. Given the teaching of this specification and as describedelsewhere herein, it is within the skill of a person skilled in the artto determine, whether the use of a monovalent antibody, such as amonovalent antibody of the present invention, against such targets wouldbe expected to produce the therapeutic effect.

Accordingly, in a further aspect, the invention relates to themonovalent antibody according to the invention as described herein foruse as a medicament.

In another aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of cancer.

In another aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of an inflammatorycondition.

In another aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of an auto(immune)disorder.

In another aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of a disorderinvolving undesired angiogenesis.

In a further aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of a disease ordisorder, which disease or disorder is treatable by administration of anantibody against a certain target, wherein the involvement of immunesystem-mediated activities is not necessary or is undesirable forachieving the effects of the administration of the antibody, and whereinsaid antibody specifically binds said antigen.

In a further aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of a disease ordisorder, which disease or disorder is treatable by blocking orinhibiting a soluble antigen, wherein multimerization of said antigenmay form undesirable immune complexes, and wherein said antibodyspecifically binds said antigen.

In a further aspect, the invention relates to the monovalent antibodyaccording to the invention for use in the treatment of a disease ordisorder, which disease or disorder is treatable by blocking orinhibiting a cell membrane bound receptor, wherein said receptor may beactivated by dimerization of said receptor, and wherein said antibodyspecifically binds said receptor.

In one embodiment of any of the above treatments, the treatmentcomprises administering one or more further therapeutic agents.

Similarly, the invention relates to the use of the monovalent antibodyaccording to the invention as described herein as a medicament.

The invention also relates to a method of treating a disease or disorderas defined herein, wherein said method comprises administering to asubject in need of such treatment a therapeutically effective amount ofa monovalent antibody according the invention, a pharmaceuticalcomposition according to the invention or a nucleic acid constructaccording to the invention. In one embodiment, the treatment comprisesadministering one or more further therapeutic agents.

Furthermore, the invention relates to the use of the monovalent antibodyaccording to the invention in the preparation of a medicament for thetreatment of a disease or disorder as defined herein.

In one embodiment of the invention, the disease or disorder to betreated is treatable by interference with cell activation through FcαRI,by interference with FcαRI function, by inhibition of subsequent FcαRIactivated IgE mediated responses, or by binding of soluble FcαRI. In oneembodiment of the invention, the monovalent antibody is directed againstFcαRI and induces apoptosis of FcαRI expressing cells. In oneembodiment, such disease or disorder may for instance be allergic asthmaor other allergic diseases such as allergic rhinitis, seasonal/perennialallergies, hay fever, nasal allergies, atopic dermatitis, eczema, hives,urticaria, contact allergies, allergic conjunctivitis, ocular allergies,food and drug allergies, latex allergies, or insect allergies, or IgAnephropathy, such as IgA pemphigus. In one such embodiment, themonovalent antibody of the invention is directed at FcαRI. Suchmonovalent antibodies may also be used for in vitro or in vivo screeningfor FcαRI in sample or patient or in an immunotoxin or radiolabelapproach to treating these diseases and disorders.

In one embodiment of the invention, the disease or disorder to betreated is treatable by downregulating Fc receptor γ-chain mediatedsignaling through FcεR1 or Fcγ receptors. Monomeric binding of antibodyto FcαRI is known to effect such inhibition. Monovalent antibodies maythus be used to inhibit immune activation through a range of Fcreceptors including Fcγ, Fcα and Fcε receptors. Thus, in one embodiment,the monovalent antibody of the invention may bind an Fcα, Fcε or Fcγreceptor, such as CD32b.

In one such embodiment, the monovalent antibody of the invention isdirected at CD25. Such monovalent antibodies may also be used for invitro or in vivo screening for CD25 in sample or patient or in animmunotoxin or radiolabel approach to treating these diseases anddisorders.

In one embodiment of the invention, the disease or disorder to betreated is treatable by antagonizing and/or inhibiting IL-15 or IL15receptor functions. In one embodiment, such disease or disorder may forinstance be arthritides, gout, connective, neurological,gastrointestinal, hepatic, allergic, hematologic, skin, pulmonary,malignant, endocrinological, vascular, infectious, kidney, cardiac,circulatory, metabolic, bone, and muscle disorders. In one suchembodiment, the monovalent antibody of the invention is directed atIL-15. Such monovalent antibodies may also be used for in vitro or invivo screening for IL-15 in a sample or patient or in an immunotoxin orradiolabel approach to treating these diseases and disorders.

In one embodiment of the invention, the disease or disorder to betreated is treatable by interfering with CD20 activity, by depleting Bcells, interfering with B cell growth and/or proliferation through forinstance an immunotoxin or radiolabel approach. In one embodiment, suchdisease or disorder may for instance be rheumatoid arthritis,(auto)immune and inflammatory disorders (as described above for IL-8related diseases and disorders), non-Hodgkin's lymphoma, B-CLL, lymphoidneoplasms, malignancies and hematological disorders, infectious diseasesand connective, neurological, gastrointestinal, hepatic, allergic,hematologic, skin, pulmonary, malignant, endocrinological, vascular,infectious, kidney, cardiac, circulatory, metabolic, bone and muscledisorders, and immune mediated cytopenia.

In one such embodiment, the monovalent antibody of the invention isdirected at CD20. Such monovalent antibodies may also be used for invitro or in vivo screening for CD20 in a sample or patient.

In one embodiment of the invention, the disease or disorder to betreated is treatable by interfering with CD38 activity, by depletingCD38 expressing cells, interfering with CD38⁺ cell growth and/orproliferation through for instance an immunotoxin or radiolabelapproach.

In one embodiment of the invention, the disease or disorder to betreated is treatable by blocking ligand-EGFr interaction, blocking EGFrfunction, depletion of EGFr expressing cells/interference with EGFr+cell growth and/or proliferation through for instance an immunotoxin orradiolabel approach.

In one such embodiment, the monovalent antibody of the invention isdirected at EGFr. Such monovalent antibodies may also be used for invitro or in vivo screening for EGFr in a sample or patient.

In one embodiment of the invention, the disease or disorder to betreated is treatable by interfering with CD4 function, depletion of CD4expressing cells/interference with CD4+ cell growth and/or proliferationthrough for instance an immunotoxin or radiolabel approach. In oneembodiment, such disease or disorder may for instance be rheumatoidarthritis, (auto)immune and inflammatory disorders (as described abovefor IL-8 related diseases and disorders), cutaneous T cell lymphomas,non-cutaneous T cell lymphomas, lymphoid neoplasms, malignancies andhematological disorders, infectious diseases, and connective,neurological, gastrointestinal, hepatic, allergic, hematologic, skin,pulmonary, malignant, endocrinological, vascular, infectious, kidney,cardiac, circulatory, metabolic, bone, and muscle disorders, and immunemediated cytopenia.

In one such embodiment, the monovalent antibody of the invention isdirected at CD4. Such monovalent antibodies may also be used for invitro or in vivo screening for CD4 in a sample or patient.

In one embodiment of the invention, a monovalent antibody directed atCD4 is used for treatment of HIV infection, or for the treatment ofAIDS.

In one embodiment of the invention, the monovalent antibodies of theinvention are monovalent antibodies of the CD4 antibodies disclosed inWO97/13852.

In one embodiment of the invention, the disease or disorder to betreated is treatable by antagonizing and/or inhibiting CD28 functions,such as preventing of co-stimulatory signals needed in T cellactivation. In one embodiment, such disease or disorder may for instancebe an inflammatory, autoimmune and immune disorder as indicated above.In one such embodiment, the monovalent antibody of the invention isdirected at CD28.

In one embodiment of the invention, the disease or disorder to betreated is treatable by altering Tissue Factor functions, such asaltering coagulation or inhibition of tissue factor signalling. In oneembodiment, such disease or disorder may for instance be vasculardiseases, such as myocardial vascular disease, cerebral vasculardisease, retinopathia and macular degeneration, and inflammatorydisorders as indicated above.

In one embodiment of the invention, the monovalent antibodies aredirected at Tissue factor, or at a complex of Factor VII and TissueFactor.

In one embodiment of the invention, the disease or disorder to betreated is treatable by interfering with Hepatitis C Virus (HCV)infection. In one such embodiment, the monovalent antibody of theinvention is directed at HCV or an HCV receptor such as CD81.

In one embodiment of the invention, the monovalent antibody is amonovalent antibody according to the invention of an antibody asdisclosed in WO2000/05266.

In one embodiment of the invention, the disease or disorder to betreated is treatable by prevention of binding of allergen toIgE-sensitized on mast cell. In one embodiment, such disease or disordermay for instance be allergen-immunotherapy of allergic diseases such asasthma, allergic rhinitis, seasonal/perennial allergies, hay fever,nasal allergies, atopic dermatitis, eczema, hives, urticaria, contactallergies, allergic conjunctivitis, ocular allergies, food and drugallergies, latex allergies, and insect allergies.

In one such embodiment, the monovalent antibody(s) of the invention areIgG4 hingeless antibodies directed towards allergen(s).

In certain embodiments, an immunoconjugate comprising a monovalentantibody conjugated with a cytotoxic agent is administered to thepatient. In some embodiments, the immunoconjugate and/or antigen towhich it is bound is/are internalized by the cell, resulting inincreased therapeutic efficacy of the immunoconjugate in killing thetarget cell to which it binds. In one embodiment, the cytotoxic agenttargets or interferes with nucleic acid in the target cell.

Examples of such cytotoxic agents include any of the chemotherapeuticagents noted herein (such as a maytansinoid or a calicheamicin), aradioactive isotope, or a ribonuclease or a DNA endonuclease.

Monovalent antibodies of the invention may be used either alone or incombination with other compositions in a therapy. For instance, amonovalent antibody of the invention may be co-administered with one ormore other antibodies, such as monovalent antibodies of the presentinvention, one or more chemotherapeutic agent(s) (including cocktails ofchemotherapeutic agents), one or more other cytotoxic agent(s), one ormore anti-angiogenic agent(s), one or more cytokines, one or more growthinhibitory agent(s), one or more anti-inflammatory agent(s), one or moredisease modifying antirheumatic drug(s) (DMARD), or one or moreimmunosuppressive agent(s), depending on the disease or condition to betreated. Where a monovalent antibody of the invention inhibits tumorgrowth, it may be particularly desirable to combine it with one or moreother therapeutic agent(s) which also inhibits tumor growth. Forinstance, anti-VEGF antibodies blocking VEGF activities may be combinedwith anti-ErbB antibodies (for instance Trastuzumab (Herceptin), ananti-HER2 antibody) in a treatment of metastatic breast cancer.Alternatively, or additionally, the patient may receive combinedradiation therapy (for instance external beam irradiation or therapywith a radioactive labeled agent, such as an antibody). Such combinedtherapies noted above include combined administration (where the two ormore agents are included in the same or separate formulations), andseparate administration, in which case, administration of the antibodyof the invention may occur prior to, and/or following, administration ofthe adjunct therapy or therapies.

In one embodiment, the monovalent antibody of the invention is amonovalent form of trastuzumab, for treatment of Her2 positive cancer.

For the prevention or treatment of disease, the appropriate dosage of amonovalent antibody of the invention (when used alone or in combinationwith other agents such as chemotherapeutic agents) will depend on thetype of disease to be treated, the type of antibody, the severity andcourse of the disease, whether the monovalent antibody is administeredfor preventive, therapeutic or diagnostic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The monovalent antibody may besuitably administered to the patient at one time or over a series oftreatments.

Such dosages may be administered intermittently, for instance every weekor every three weeks (for instance such that the patient receives fromabout two to about twenty, for instance about six doses of themonovalent antibody). An initial higher loading dose, followed by one ormore lower doses may be administered. An exemplary dosing regimencomprises administering an initial loading dose of about 4 mg/kg,followed by a weekly maintenance dose of about 2 mg/kg of the monovalentantibody. However, other dosage regimens may be useful. In oneembodiment, the monovalent antibodies of the invention are administeredin a weekly dosage of from 50 mg to 4000 mg, for instance of from 250 mgto 2000 mg, such as for example 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mgor 2000 mg, for up to 8 times, such as from 4 to 6 times. The weeklydosage may be divided into two or three subdosages and administered overmore than one day. For example, a dosage of 300 mg may be administeredover 2 days with 100 mg on day one (1), and 200 mg on day two (2). Adosage of 500 mg may be administered over 3 days with 100 mg on day one(1), 200 mg on day two (2), and 200 mg on day three (3), and a dosage of700 mg may be administered over 3 days with 100 mg on day 1 (one), 300mg on day 2 (two), and 300 mg on day 3 (three). The regimen may berepeated one or more times as necessary, for example, after 6 months or12 months.

The dosage may be determined or adjusted by measuring the amount ofcirculating monovalent antibodies of the invention upon administrationin a biological sample for instance by using anti-idiotypic antibodieswhich target said monovalent antibodies.

In one embodiment, the monovalent antibodies of the invention may beadministered by maintenance therapy, such as, for instance once a weekfor a period of 6 months or more.

In one embodiment, the monovalent antibodies of the invention may beadministered by a regimen including one infusion of a monovalentantibody of the invention followed by an infusion of same monovalentantibody conjugated to a radioisotope. The regimen may be repeated, forinstance 7 to 9 days later.

In another main aspect, the invention relates to the use of a monovalentantibody according to the invention as a diagnostic agent.

As described above, in a further aspect, the invention relates to astabilized IgG4 antibody for use as a medicament, comprising a heavychain and a light chain, wherein said heavy chain comprises a human IgG4constant region having the sequence set forth in SEQ ID NO:2, whereinLys (K) in position 250 has been replaced by Gln (Q) or Glu (E) andwherein the antibody optionally comprises one or more furthersubstitutions, deletions and/or insertions in the constant region as setforth in SEQ ID NO:2.

In one embodiment thereof, the human IgG4 constant region has thesequence set forth in SEQ ID NO:2, wherein X1 at position 189 is Leu andX2 at position 289 is Arg. In another embodiment thereof, the human IgG4constant region has the sequence set forth in SEQ ID NO:2, wherein X1 atposition 189 is Leu and X2 at position 289 is Lys. In yet anotherembodiment thereof, the human IgG4 constant region has the sequence setforth in SEQ ID NO:2, wherein X1 at position 189 is Val and X2 atposition 289 is Arg.

In one further aspect, the invention relates to an isolated stabilizedIgG4 antibody for use as a medicament, comprising a heavy chain and alight chain, wherein said heavy chain comprises a human IgG4 constantregion having the sequence set forth in SEQ ID NO:2, wherein Lys (K) inposition 250 has been replaced by Gln (Q) or Glu (E) and wherein theantibody optionally comprises one or more further substitutions,deletions and/or insertions in the constant region as set forth in SEQID NO:2.

In one embodiment thereof, the human IgG4 constant region has thesequence set forth in SEQ ID NO:2, wherein X1 at position 189 is Leu andX2 at position 289 is Arg. In another embodiment thereof, the human IgG4constant region has the sequence set forth in SEQ ID NO:2, wherein X1 atposition 189 is Leu and X2 at position 289 is Lys. In yet anotherembodiment thereof, the human IgG4 constant region has the sequence setforth in SEQ ID NO:2, wherein X1 at position 189 is Val and X2 atposition 289 is Arg.

The stabilized IgG4 antibodies according to the invention have theadvantage that they contain a minimal number of sequence changes in theconstant region as compared to naturally occurring IgG4. This reducesthe risk of immunogenicity when the antibody is used for human therapy.

In one embodiment thereof the stabilized IgG4 antibody does not comprisea Cys-Pro-Pro-Cys sequence in the hinge region.

In one embodiment thereof the CH3 region of the stabilized IgG4 antibodyhas been replaced by the CH3 region of human IgG1, of human IgG2 or ofhuman IgG3.

In one embodiment thereof the stabilized IgG4 antibody does not comprisea substitution of the Leu (L) residue at the position corresponding to115 by a Glu (E).

In one embodiment thereof the stabilized IgG4 antibody does comprise asubstitution of the Leu (L) residue at the position corresponding to 115by a Glu (E).

In one embodiment thereof the stabilized IgG4 antibody comprises one ormore of the following substitutions an Ala (A) at position 114, an Ala(A) at position 116, an Ala (A) at position 117, an Ala (A) at position177, an Ala (A) or Val (V) at position 198, an Ala (A) at position 200,an Ala (A) or Gln (Q) at position 202.

In one embodiment thereof the stabilized IgG4 antibody comprises a CXPCor CPXC sequence in the hinge region, wherein X can be any amino acidexcept for Pro (P).

In one embodiment thereof the stabilized IgG4 antibody does not comprisean extended IgG3-like hinge region, such as the extended hinge region asset forth in FIG. 14.

In one embodiment thereof the stabilized IgG4 antibody comprises a CPSCsequence in the hinge region.

In one embodiment thereof the stabilized IgG4 antibody has less than 25,such as less than 10, e.g. less than 9, 8, 7, 6, 5, 4, 3, or 2substitutions, deletions and/or insertions in the constant region as setforth in SEQ ID NO:2.

Typically, the stabilized IgG4 antibody of the invention has a lowerability to activate effector functions as compared to IgG1 and IgG3. Inone embodiment thereof the antibody is less efficient in mediating CDCand/or ADCC than a corresponding IgG1 or IgG3 antibody having the samevariable regions. Assays for measuring CDC or ADCC activity are wellknown in the art.

In one embodiment thereof the stabilized IgG4 antibody is selected fromthe group consisting of a human monoclonal antibody, a humanizedmonoclonal antibody and a chimeric monoclonal antibody.

In one embodiment thereof the stabilized IgG4 antibody comprises a humankappa light chain.

In one embodiment thereof the stabilized IgG4 antibody comprises a humanlambda light chain.

In one embodiment thereof the stabilized IgG4 antibody is a bivalentantibody, for example an antibody which is bivalent even in the presenceof excess of irrelevant antibodies, as explained in the Examples herein.

In one embodiment thereof the stabilized IgG4 antibody is a full-lengthantibody.

Methods for the production of stabilized IgG4 antibodies are well-knownin the art. In a preferred embodiment, antibodies of the invention aremonoclonal antibodies. Monoclonal antibodies may e.g. be produced by thehybridoma method first described by Kohler et al., Nature 256, 495(1975), or may be produced by recombinant DNA methods. Monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in, for example, Clackson et al., Nature 352,624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991).Monoclonal antibodies may be obtained from any suitable source. Thus,for example, monoclonal antibodies may be obtained from hybridomasprepared from murine splenic B cells obtained from mice immunized withan antigen of interest, for instance in form of cells expressing theantigen on the surface, or a nucleic acid encoding an antigen ofinterest. Monoclonal antibodies may also be obtained from hybridomasderived from antibody-expressing cells of immunized humans or non-humanmammals such as rats, dogs, primates, etc.

Further modifications, such as amino acid substitutions, deletions orinsertion as described above, may be performed using standardrecombinant DNA techniques well-known in the art.

In one embodiment, the stabilized IgG4 antibody of the invention is ahuman antibody.

In a further main aspect, the invention relates to a method forproducing a stabilized IgG4 antibody of the invention, said methodcomprising expressing a nucleic acid construct encoding said antibody ina host cell and optionally purifying said antibody.

In one embodiment, the stabilized IgG4 antibody of the invention islinked to a compound selected from the group consisting of a cytotoxicagent; a radioisotope; a prodrug or drug, such as a taxane; a cytokine;and a chemokine. Methods for linking (conjugating) such compounds to anantibody are well-known in the art. References to suitable methods havebeen given in WO 2004/056847 (Genmab).

In one embodiment thereof the stabilized IgG4 antibody is linked to acompound selected from the group consisting of a cytotoxic agent; aradioisotope; a prodrug or drug, such as a taxane; a cytokine; and achemokine.

In a further main aspect, the invention relates to a pharmaceuticalcomposition comprising a stabilized IgG4 antibody as defined hereinabove. The pharmaceutical compositions may be formulated withpharmaceutically acceptable carriers or diluents as well as any otherknown adjuvants and excipients in accordance with conventionaltechniques, such as those disclosed in Remington: The Science andPractice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co.,Easton, Pa., 1995.

In one embodiment, a pharmaceutical composition of the present inventionis administered parenterally. The phrases “parenteral administration”and “administered parenterally” as used herein means modes ofadministration other than enteral and topical administration, usually byinjection, and include epidermal, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, intratendinous, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, intracranial, intrathoracic, epidural and intrasternalinjection and infusion.

The stabilized IgG4 antibodies of the invention can be used in thetreatment and/or prevention of a number of diseases, and be directed toan antigen selected from a broad variety of suitable target molecules.

In one embodiment thereof the stabilized IgG4 antibody according to anyone of the above embodiments binds to an antigen selected from the groupconsisting of erythropoietin, beta-amyloid, thrombopoietin,interferon-alpha (2a and 2b), interferon-beta (1b), interferon-gamma,TNFR I (CD120a), TNFR II (CD120b), IL-1R type 1 (CD121a), IL-1R type 2(CD121b), IL-2, IL2R (CD25), IL-2R-beta (CD123), IL-3, IL-4, IL-3R(CD123), IL-4R (CD124), IL-5R (CD125), IL-6R-alpha (CD126), -beta(CD130), IL-8, IL-10, IL-11, IL-15, IL-15BP, IL-15R, IL-20, IL-21, TCRvariable chain, RANK, RANK-L, CTLA4, CXCR4R, CCR5R, TGF-beta1, -beta2,-beta3, G-CSF, GM-CSF, MIF-R (CD74), M-CSF-R (CD115), GM-CSFR (CD116),soluble FcRI, sFcRII, sFcRIII, FcRn, Factor VII, Factor VIII, Factor IX,VEGF, VEGFxxxb, alpha-4 integrin, Cd11a, CD18, CD20, CD38, CD25, CD74,FcepsilonRI, acetyl choline receptor, fas, fasL, TRAIL, hepatitis virus,hepatitis C virus, envelope E2 of hepatitis C virus, tissue factor, acomplex of tissue factor and Factor VII, EGFr, CD4, CD28, VLA-1,2,3, or4, LFA-1, MAC-1, I-selectin, PSGL-1, ICAM-1, P-selectin, periostin, CD33(Siglec 3), Siglec 8, TNF, CCL1, CCL2, CCL3, CCL4, CCL5, CCL11, CCL13,CCL17, CCL18, CCL20, CCL22, CCL26, CCL27, CX3CL1, LIGHT, EGF, VEGF,TGFalpha, HGF, PDGF, NGF, complement or a related components such as:C1q, C4, C2, C3, C5, C6, C7, C8, C9, MBL, factor B, a Matrix MetalloProtease such as any of MMP1 to MMP28, CD32b, CD200, CD200R, KillerImmunoglobulin-Like Receptors (KIRs), NKG2D and related molecules,leukocyte-associated immunoglobulin-like receptors (LAIRs), ly49, PD-L2,CD26, BST-2, ML-IAP (melanoma inhibitor of apoptosis protein), cathepsinD, CD40, CD40R, CD86, a B cell receptor, CD79, PD-1 and a T cellreceptor.

In one embodiment thereof

(i) the antibody binds to an alpha-4 integrin and is for use in thetreatment of inflammatory and autoimmune diseases, such as rheumatoidarthritis, multiple sclerosis, inflammatory bowel disease, asthma andsepsis;(ii) the antibody binds to VLA-1,2,3, or 4 and is for use in thetreatment of inflammatory and autoimmune diseases, such as rheumatoidarthritis, multiple sclerosis, inflammatory bowel disease, asthma,type-1 diabetes, SLE, psoriasis, atopic dermatitis, COPD and sepsis;(iii) the antibody binds to a molecule selected from the groupconsisting of LFA-1, MAC-1, I-selectin and PSGL-1 and is for use in thetreatment of inflammatory and autoimmune diseases, such as rheumatoidarthritis, multiple sclerosis, inflammatory bowel disease, asthma,type-1 diabetes, SLE, psoriasis, atopic dermatitis, and COPD;(iv) the antibody binds to a molecule selected from the group consistingof LFA-1, MAC-1, I-selectin and PSGL-1 and is for use in the treatmentof a disease selected from the group consisting of ischemia-reperfusioninjury, cystic fibrosis, osteomyelitis, glomerulonepritis, gout andsepsis;(v) the antibody binds to CD18 and is for use in the treatment ofinflammatory and autoimmune diseases, such as rheumatoid arthritis,multiple sclerosis, inflammatory bowel disease, asthma, type-1 diabetes,SLE, psoriasis, atopic dermatitis and COPD;(vi) the antibody binds to Cd11a and is for use in the treatment ofinflammatory and autoimmune diseases, such as rheumatoid arthritis,multiple sclerosis, inflammatory bowel disease, asthma, type-1 diabetes,SLE, psoriasis, atopic dermatitis and COPD;(vii) the antibody binds ICAM-1 and is for use in the treatment ofinflammatory and autoimmune diseases, such as rheumatoid arthritis,multiple sclerosis, inflammatory bowel disease, asthma, type-1 diabetes,SLE, psoriasis, atopic dermatitis and COPD;(viii) the antibody binds to P-selectin and is for use in the treatmentof cardiovascular diseases, post-thrombotic vein wall fibrosis, ischemiareperfusion injury, inflammatory diseases or sepsis;(ix) the antibody binds to periostin and is for use in the treatment ofmalignant diseases and/or metastasizing diseases, such as ovary cancer,endometrial cancer, NSCLC, glioblastoma, brain-related tumors, breastcancer, OSCC, colon cancer, pancreatic cancer, HNSCC, kidney cancer,thymoma, lung cancer, skin cancer, larynx cancer, liver cancer, parotidtumors, gastric cancer, esophagus cancer, prostate cancer, bladdercancer and cancer of the testis;(x) the antibody binds to CD33 (Siglec 3), is optionally coupled to atoxin, cytotoxic or cytostatic drug, and is for use in the treatment oftumors expressing CD33 or acute myeloid leukemia;(xi) the antibody binds to Siglec 8 and is for use in the treatment ofasthma, inflammatory or autoimmune diseases, such as rheumatoidarthritis, multiple sclerosis, inflammatory bowel disease, asthma,type-1 diabetes, SLE, psoriasis, atopic dermatitis and COPD;(xii) the antibody binds to TNF and is for use in the treatment ofinflammatory and autoimmune diseases, such as rheumatoid arthritis,multiple sclerosis, inflammatory bowel disease, asthma, type-1 diabetes,SLE, psoriasis, atopic dermatitis, COPD and sepsis;(xiii) the antibody binds to CCL1, CCL2, CCL3, CCL4, CCL5, CCL11, CCL13,CCL17, CCL18, CCL20, CCL22, CCL26, CCL27 or CX3CL1 and is for use in thetreatment of atopic dermatitis, inflammatory and autoimmune diseases,such as rheumatoid arthritis, multiple sclerosis, inflammatory boweldisease, asthma, type-1 diabetes, SLE, psoriasis, COPD and sepsis;(xiv) the antibody binds to LIGHT and is for use in the treatment of adisease selected from the group consisting of: hepatitis, inflammatorybowel disease, GVHD and inflammation;(xv) the antibody binds to EGF, VEGF, TGFalpha or HGF and is for use inthe treatment of: malignant diseases, such as solid cancers;(xvi) the antibody binds to PDGF and is for use in the treatment ofdiseases in which abnormal cell proliferation cell migration and/orangiogenesis occurs, such as atherosclerosis, fibrosis, and malignantdiseases;(xvii) the antibody binds to NGF and is for use in the treatment ofneurological diseases, neurodegenerative diseases, such as Alzheimer'sdisease and Parkinson's disease, or cancer, such as prostate cancer;(xviii) the antibody binds to complement or a related components such asC1q, C4, C2, C3, C5, C6, C7, C8, C9, MBL, or factor B and is for use indiseases in which complement and related components play a detrimentalrole, such as organ transplant rejection, multiple sclerosis,Guillain-Barré syndrome, hemolytic anemia, Paroxysmal NocturnalHemoglobinuria, stroke, heart attacks, burn injuries, age-relatedmacular degeneration, asthma, lupus, arthritis, myasthenia gravis,anti-phospholipid syndrome, sepsis and ischemia reperfusion injury;(xix) the antibody binds to a Matrix Metallo Protease such as any ofMMP1 to MMP28 and is for use in the treatment of inflammatory andautoimmune diseases, cancer, including metastatic cancer; arthritis,inflammation, cardiovascular diseases, cerebrovascular diseases such asstroke or cerebral aneurysms, pulmonary diseases such as asthma, oculardiseases such as corneal wound healing or degenerative genetic eyediseases, gastrointestinal diseases such as inflammatory bowel diseaseor ulcers, oral diseases such as dental caries, oral cancer orperiodontitis, ischemia reperfusion injury or sepsis;(xx) the antibody binds to CD32b and is for use in enhancement of T-cellresponses to tumor antigens and ADCC/phagocytosis by macrophages, incombination with another therapeutic antibody; vaccination,immunotherapy of B-cell lymphoma's, asthma or allergy;(xxi) the antibody binds to CD200 or CD200R and is for use in thetreatment of: asthma, rheumatoid arthritis, GVHD, other autoimmunediseases, or cancer, such as solid tumors or lymphomas;(xxii) the antibody binds to Killer Immunoglobulin-Like Receptors(KIRs), NKG2D or related molecules, leukocyte-associatedimmunoglobulin-like receptors (LAIRs), or ly49 and is for use in thetreatment of: cancer, such as solid tumors or lymphomas; asthma,rheumatoid arthritis, GVHD or other autoimmune diseases;(xxiii) the antibody binds to PD-L2 and is for use in the treatment ofcancer, asthma, or for use in vaccine enhancement;(xxiv) the antibody binds to CD26 and is for use in the treatment of:atherosclerosis, GVHD, or auto-immune diseases;(xxv) the antibody binds to BST-2 and is for use in the treatment ofasthma, atherosclerosis, rheumatoid arthritis, psoriasis, Crohn'sdisease, ulcerative cholitis, atopic dermatitis, sepsis or inflammation;(xxvi) the antibody binds to ML-IAP (melanoma inhibitor of apoptosisprotein) and is for use in the treatment of melanoma;(xxvii) the antibody binds to cathepsin D and is for use in thetreatment of malignant diseases such as breast cancer, ovarian cancer,glioma, NSCLC, bladder cancer, endometrial cancer, liver cancer,sarcoma, gastric cancer, SCCHN, prostate cancer or colorectal cancer;(xxviii) the antibody binds to CD40 or CD40R and is for use in thetreatment of cancer, in particular B-cell lymphomas, B-cell-related or-mediated diseases, autoimmune diseases such as psoriatic arthritis,rheumatoid arthritis, multiple sclerosis, psoriasis, Crohn's disease orulcerative cholitis;(xxix) the antibody binds to CD86 and is for use in conjunction withorgan transplantation;(xxx) the antibody binds to a B cell receptor and is for use in thetreatment of: B-cell-related or -mediated diseases, such as B celllymphoma's, leukemia, autoimmune diseases, inflammation or allergy;(xxxi) the antibody binds to CD79 and is for use in the treatment ofB-cell-related or -mediated diseases, such as B-cell lymphomas,leukemia, autoimmune diseases, inflammation or allergy;(xxxii) the antibody binds to a T cell receptor and is for use in thetreatment of T-cell-related or -mediated diseases, such as T-celllymphomas, leukemia, autoimmune diseases, inflammation or allergy;(xxxiii) the antibody binds to FcalphaRI and is for use in the treatmentof a disease or disorder selected from allergic asthma or other allergicdiseases such as allergic rhinitis, seasonal/perennial allergies, hayfever, nasal allergies, atopic dermatitis, eczema, hives, urticaria,contact allergies, allergic conjunctivitis, ocular allergies, food anddrug allergies, latex allergies, or insect allergies, or IgAnephropathy, such as IgA pemphigus;(xxxiv) the antibody binds to CD25 and is for use in the treatment of adisease or disorder selected from the group consisting of transplantrejection, graft-versus-host disease, inflammatory, immune or autoimmunediseases, inflammatory or hyperproliferative skin disorders, lymphoidneoplasms, malignancies, hematological disorders, skin disorders,hepato-gastrointestinal disorders, cardiac disorders, vasculardisorders, renal disorders, pulmonary disorders, neurological disorders,connective tissue disorders, endocrinological disorders, and viralinfections;(xxxv) the antibody binds to IL-15 or the IL15 receptor and is for usein the treatment of a disease or disorder selected from the groupconsisting of: arthritides, gout, connective disorders, neurologicaldisorders, gastrointestinal disorders, hepatic disorders, allergicdisorders, hematologic disorders, skin disorders, pulmonary disorders,malignant disorders, endocrinological disorders, vascular disorders,infectious disorders, kidney disorders, cardiac disorders, circulatorydisorders, metabolic disorders, bone, disorders and muscle disorders;(xxxvi) the antibody binds to IL-8 and is for use in the treatment of adisease or disorder selected from the group consisting of palmoplantarpustulosis (PPP), psoriasis, or other skin diseases, inflammatory,autoimmune and immune disorders, alcoholic hepatitis and acutepancreatitis, diseases involving IL-8 mediated angiogenesis;(xxxvii) the antibody binds to CD20 and is for use in the treatment of adisease or disorder selected from the group consisting of: rheumatoidarthritis, (auto)immune and inflammatory disorders, non-Hodgkin'slymphoma, B-CLL, lymphoid neoplasms, malignancies and hematologicaldisorders, infectious diseases and connective disorders, neurologicaldisorders, gastrointestinal disorders, hepatic disorders, allergicdisorders, hematologic disorders, skin disorders, pulmonary disorders,malignant disorders, endocrinological disorders, vascular disorders,infectious disorders, kidney disorders, cardiac disorders, circulatorydisorders, metabolic disorders, bone and muscle disorders, and immunemediated cytopenia;(xxxviii) the antibody binds to CD38 and is for use in the treatment ofa disease or disorder selected from the group consisting of tumorigenicdisorders, immune disorders in which CD38 expressing B cells, plasmacells, monocytes and T cells are involved, acute respiratory distresssyndrome and choreoretinitis, rheumatoid arthritis, inflammatory, immuneand/or autoimmune disorders in which autoantibodies and/or excessive Band T lymphocyte activity are prominent, skin disorders, immune-mediatedcytopenias, connective tissue disorders, arthritides, hematologicdisorders, endocrinopathies, hepato-gastrointestinal disorders,nephropathies, neurological disorders, cardiac and pulmonary disorders,allergic disorders, ophthalmologic disorders, infectious diseases,gynecological-obstetrical disorders, male reproductive disorders,transplantation-derived disorders;(xxxix) the antibody binds to EGFr and is for use in the treatment of adisease or disorder selected from the group consisting of: cancers(over)expressing EGFr and other EGFr related diseases, such asautoimmune diseases, psoriasis, and inflammatory arthritis;(xxxx) the antibody binds to CD4 and is for use in the treatment of adisease or disorder selected from the group consisting of rheumatoidarthritis, (auto)immune and inflammatory disorders, cutaneous T celllymphomas, non-cutaneous T cell lymphomas, lymphoid neoplasms,malignancies and hematological disorders, infectious diseases, andconnective disorders, neurological disorders, gastrointestinaldisorders, hepatic disorders, allergic disorders, hematologic disorders,skin disorders, pulmonary disorders, malignant disorders,endocrinological disorders, vascular disorders, infectious disorders,kidney disorders, cardiac disorders, circulatory disorders, metabolicdisorders, bone disorders, muscle disorders, immune mediated cytopenia,and HIV infection/AIDS;(xxxxi) the antibody binds CD28 and is for use in the treatment of adisease or disorder selected from the group consisting of aninflammatory disease, autoimmune disease and immune disorder;(xxxxii) the antibody binds to tissue factor, or a complex of Factor VIIand tissue factor and is for use in the treatment of a disease ordisorder selected from the group consisting of vascular diseases, suchas myocardial vascular disease, cerebral vascular disease, retinopathyand macular degeneration, and inflammatory disorders; or(xxxxiii) the antibody binds to PD-1 and is for use in the treatment ofHIV-1/AIDS.

In a further embodiment the invention relates to a pharmaceuticalcomposition, characterized in that it comprises a stabilized IgG4antibody as defined in any one of the above embodiments and apharmaceutically acceptable carrier or excipient.

In a further embodiment the invention relates to the use of a stabilizedIgG4 antibody according to any one of the above embodiments (i) to(xxxxiii) for the preparation of a medicament for the treatment of adisease as specified in any one of the above related embodiments (i) to(xxxxiii).

In a further embodiment the invention relates to a method for thetreatment of a subject suffering from a disease as specified in any oneof the above embodiments (i) to (xxxxiii) comprising administering tothe subject in need thereof a stabilized IgG4 antibody according to asspecified in any one of the above related embodiments (i) to (xxxxiii).

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting.

EXAMPLES Example 1 Structural Analysis of CH3-CH3 Interface

In human IgG1, the non-covalent interaction between the CH3 domainsinvolves 16 residues located on four anti-parallel β-strands that makeintermolecular contacts and burry 1090 Å² from each surface(Deisenhofer, J.; Biochemistry, 1981. 20(9): p. 2361-70). Alaninescanning mutagenesis showed that stabilization of the IgG1 CH3-CH3interaction was largely mediated by 6 of these residues, including K409(Dall'Acqua, W., et al.; Biochemistry, 1998. 37(26): p. 9266-73). To geta better understanding of the role of K409 in the IgG1 CH3-CH3interaction, the 1.65 Å1 L6X crystal structure (Idusogie, E. E., et al.;J Immunol, 2000. 164(8): p. 4178-84) was studied in more detail usingthe Brugel modelling package (Delhaise, P., et al., J. Mol. Graph.,1984. 2(4): p. 103-106).

In order to propose mutations that should lead to a desiredstabilization (or destabilization) of IgG4, a quantitativestructure-based scoring methodology was employed (Desmet, J., et al.,;Proteins, 2005. 58(1): p. 53-69). Briefly, each position in the CH3-CH3dimer interface was subjected to mutagenesis to all natural amino acids,except cysteine and proline. Subsequent to mutagenesis, Exploration ofthe conformational space was obtained by interdependent optimization ofthe side chains of all residues located in a sphere of 12 Å of themutated residue, using the FASTER algorithm (Desmet, J., et al.,;Proteins, 2002. 48(1): p. 31-43), performed on all macro-rotamericstates for the side chain under investigation. Subsequently, on eachmacro-rotameric state thus obtained, a scoring function for the sidechain under investigation was evaluated, as described (Desmet, J., etal.,; Proteins, 2005. 58(1): p. 53-69). Finally, per position in theCH3-CH3 dimer interface, the highest scores for each mutation werecompared, and visual inspection of the resulting conformation wascarried out in selected cases.

Example 2 Water Hypothesis

In the IgG1 structure, K409 forms a hydrogen bond with D399′ on theopposite CH3 domain. Furthermore, K409 is part of a water-binding pockettogether with S364 and T411 in the same CH3 domain and K370′ on theopposite CH3 domain. The presence of the water molecule prevents anelectrostatic clash between K409 and K370′.

The K409R substitution (as in IgG4) was modelled in the 1 L6X structureby optimizing the side chain conformations of the arginine residue andits surrounding residues, using the FASTER algorithm (Desmet, J., etal.,; Proteins, 2002. 48(1): p. 31-43). In this model, the guanidiniumgroup of R409 takes up the position of the water molecule and causes anelectrostatic clash with K370′. The side-chains of T411 and K370′ loosetheir interactions compared to the case with water present (as in IgG1),but D399 keeps its interaction with the side chain at position R409.

Example 3 Destabilization of IgG4

The mutations in the Table below were made in order to destabilize theCH3—CH3 interaction of an IgG4.

KABAT indicates amino acid numbering according to Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). EU indexindicates amino acid numbering according to EU index as outlined inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)).

Numbering of CH3 mutations KABAT EU index G4 SEQ ID NO: 4 370 Y349R*Y217R* 372 L351N* L219N* 372 L351Q* L219Q* 378 E357A E225A 378 E357T*E225T* 378 E357V* E225V* 378 E357I* E225I* 387 S364R* S232R* 387 S364K*S232K* 389 T366A T234A 389 T366R* T234R* 389 T366K* T234K* 389 T366N*T234N* 391 L368A L236A 391 L368V L236V 391 L368E* L236E* 391 L368G*L236G* 391 L368S* L236S* 391 L368T* L236T* 393 K370A K238A 393 K370R*K238R* 393 K370T K238T 427 D399A D267A 427 D399T* D267T* 427 D399S*D267S* 436 F405A F273A 436 F405L F273L 436 F405T* F273T* 436 F405D*F273D* 436 F405R* F273R* 436 F405Q* F273Q* 436 F405K* F273K* 436 F405YF273Y 438 Y407A Y275A 438 Y407E* Y275E* 438 Y407Q* Y275Q* 438 Y407K*Y275K* 438 Y407F Y275F 440 R409A R277A 440 R409K R277K (stabilizing seeWO2008145142) 440 R409E* R277E* 442 T411D* T279D* 442 T411V* T279V* 442T411N* T279N*

Example 4 Various Technical Procedures

The following techniques were performed as described in WO2007059782:Oligonucleotide primers and PCR amplification, agarose gelelectrophoresis, analysis and purification of PCR products and enzymaticdigestion products, quantification of DNA by UV spectroscopy,restriction enzyme digestions, ligation of DNA fragments, transformationof E. coli, screening of bacterial colonies by PCR, plasmid DNAisolation from E. coli culture, site-directed mutagenesis, DNAsequencing and transient expression in HEK-293F cells.

Example 5 Constructions and Biochemical Analysis of CH3 Variants of2F8-HG

The above-described mutations were introduced into the CH3 region ofhingeless anti-EGFR antibody 2F8-HG, described in WO2007059782. To makethe constructs for the expression of the CH3 mutants, the mutations wereintroduced into pTomG42F8HG (described in WO2007059782) usingsite-directed mutagenesis. The constructs were expressed transiently andpurified as described in WO2007059782.

In order to investigate whether CH3 variant HG molecules exist asmonomers or dimers, a mass spectrometry method was employed as describedin WO2007059782.

FIG. 1 shows a summary of the monomer/dimer ratios obtained for each HGmutant using non-covalent nano-electrospray mass spectrometry. CH3mutants showed a substantial increase in monomer/dimer ratio compared to2F8-HG (WT). The percentage molecules present as monomers increased from15% in 2F8-HG (WT) to >80% in most CH3 mutants, except for mutationR277A. HG mutation R277K, which introduces an IgG1 sequence into theIgG4 backbone, was used as negative control. As expected, this mutantbehaved as dimer.

The monomer or dimer configuration of CH3 mutants was verified usingNativePAGE™ Novex® Bis-Tris gel electrophoresis (Invitrogen, Carlsbad,Calif.) according to the instructions of the manufacturer as shown inFIG. 2. This native gel electrophoresis technique uses Coomassie G-250as a charge-shift molecule instead of SDS and is able to maintain nativeprotein conformation and protein complex quaternary structures (SchaggerH and von Jagow G 1991 Blue native gel electrophoresis for isolation ofmembrane complexes in enzymatically active form. Anal. Biochem.199:223-244).

Under these experimental conditions, 2F8-HG (WT) and R277K and R277Ashowed a protein band corresponding to the size of a full tetrameric(two heavy and two light chains) molecule. The CH3 mutants T234A, L236A,L236V, F273A, F273L, and Y275A were shown to be half molecules (only oneheavy and one light chain).

Example 6 Functional Analysis of CH3 Mutants of 2F8-HG

Binding of 2F8-HG (WT) and variants was determined in the absence andpresence of 200 μg/ml polyclonal human IgG (Intravenous Immunoglobulin,IVIG, Sanquin Netherlands) (as described in Example 57 of WO2007059782).

FIGS. 3 and 4 show that the binding curve of 2F8-HG in the presence ofIVIG clearly right-shifts with respect to the binding curve of 2F8-HGwithout IVIG. This difference in avidity for the EGFr coat is consistentwith the idea that, in the presence of IVIG, 2F8-HG binds monovalently(see Example 57 of WO2007059782). The binding curves of several of thetested mutations, 2F8-HG-T234A, 2F8-HG-L236V, 2F8-HG-L236A and2F8-HG-Y275A, become insensitive to the addition of IVIG and weresuper-imposable on the monovalent binding curve of 2F8-HG in thepresence of IVIG. These differences in avidity for the EGFr coat areconsistent with the idea that the 2F8-HG-T234A, 2F8-HG-L236V,2F8-HG-L236A and 2F8-HG-Y275A mutations prevent dimerization of the HGmolecules.

Example 7 Functional analysis of CH3 mutants of 2F8-HG

CH3 mutants of 2F8-HG were shown to bind EGFr with lower apparentaffinities than 2F8-HG in a binding ELISA coated with EGFr protein (seeabove). The potency of 2F8-HG CH3 mutants to inhibit ligand-induced EGFrphosphorylation in cells in vitro was compared to that of 2F8-HG (WT)and 2F8-Fab fragments in the Phosphorylation Inhibition Assay (PIA) asdescribed in example 54 of WO2007059782.

CH3 HG mutants were less potent to inhibit EGFr phosphorylation than2F8-HG (WT) and the control mutants R277K and R277A, in line with theincrease in monomer/dimer ratio of these mutants (FIG. 5).

Example 8 Concentration Dependent Configuration of CH3 Mutants of HG

The monomer/dimer configuration of CH3 mutants F273A, L236V, and Y275Awas further investigated at different concentrations, ranging from0.01-10 μM using non-covalent nano-electrospray mass spectrometry asdescribed in WO2007059782. The monomer/dimer configuration of these CH3mutants was compared to the configuration of 2F8-HG (WT) and R277K.

FIG. 6 shows that all HG mutants were 100% monomeric at lowconcentrations (except for R277K which behaved as dimer). With increasedconcentration of HG mutants, a decrease in monomericity was observed.However, the figure shows that the CH3 mutants exhibited such decreasein monomericity at much higher concentration than 2F8-HG (WT). Hence,the CH3 mutants contained a higher percentage of monomer molecules athigher molar concentrations.

For 2F8-HG (WT) and mutants E225A, E225V, S232R, T234A, L236A, L236T,L236V, L236E, L236S, L236G, K238A, K238T, D267S, D267A, F273A, F273L,F273Y, F273D, F273T, F273R, F273Q, Y275A, Y275Q, Y275K, Y275E, R277A,R277K, D267S+Y275E, D267S+Y275K, D267S+Y275Q, F273D+Y275E andF273T+Y275E signals corresponding to the monomeric (M_(s)) and dimeric(D_(s)) configurations were integrated and the relative proportion ofeach configuration at each concentration ([M]₀) was determined using thefollowing equations:

[M] _(eq) =M _(S)/(M _(S) +D _(S))·[M] ₀; concentration monomer atequilibrium

[D] _(eq)=([M] ₀ −[M] _(eq))/2; concentration dimer at equilibrium

Dissociation constant (K_(D)) values were subsequently calculated forall mutants by plotting the [D]_(eq) against [M]_(eq) ² values of eachconcentration and determining the gradient by least-squares linearregression using Excell software (Microsoft). The K_(D) measured for2F8-HG (WT) was 5.0×10⁻⁸ M. The relative K_(D) of each mutant comparedto the K_(D) of 2F8-HG (WT) was calculated and plotted.

FIG. 7 shows that all HG mutants (except for R277K, K238A and K238T) hada higher relative K_(D), which translates into an increase in monomericbehavior compared to 2F8-HG (WT). The R277K, K238A and K238T mutantsshowed a lower relative K_(D), meaning that they stabilize the CH3-CH3interaction.

Example 9 Removal of Glycosylation Sites

To remove (potential) acceptor sites for N-linked glycosylation(“glycosylation sites”) from the monovalent antibody, alterations to thesequence were made. To examine how this could be achieved withintroducing a minimum of T cell epitopes, and without perturbing thenative structure of the molecule, an in silico analysis was performed.The HLA binding specificities of all possible 10-mer peptides derivedfrom a target sequence were analyzed (Desmet et al. 1992, 1997, 2002,2005; Van Walle et al. 2007 Expert Opinion on Biological Therapy7:405-418). Profiling was done at the allotype level for 20 DRB1, 7DRB3/4/5, 14 DQ and 7 DP, i.e. 48 HLA class II receptors in total.Quantitative estimates of the free energy of binding deltaGbind of apeptide for each of the 48 HLA class II receptors were calculated. Thesedata were then further processed by classifying peptides as strong,medium, weak and non-binders.

The table below shows the 27 sequence variants which contain only mediumepitopes, specific for no more than three different DRB1 allotypes.

TABLE Summary of sequence variants containing either a single mediumDRB1 epitope, or multiple medium epitopes affecting three or less MHCallotypes. DRB1M DRB1*0101 DRB1*0102 DRB1*0401 DRB1*0402 DRB1*0405DRB1*0407 DRB1*0801 NST 0 DST 1 1 1 EST 1 1 1 GST 1 1 HST 1 1 1 MST 1 1PST 1 1 QST 1 1 1 1 SST 1 1 1 TST 1 1 1 CSE 2 1 1 CSP 2 1 1 DSE 2 1 1DSG 2 1 1 DSP 2 1 1 ESE 2 1 1 1 ESP 2 1 1 GSE 2 1 1 1 GSP 2 1 1 HSE 2 11 MSE 2 1 1 1 NSE 2 1 1 NSP 2 1 1 PSE 2 1 1 1 PSP 2 1 1 SSE 2 1 1 SSP 31 1 1 TSP 3 1 1 1 DRB1*0802 DRB1*0901 DRB1*1101 DRB1*1104 DRB1*1301DRB1*1401 NST DST 1 1 EST 1 GST 1 HST 1 1 MST 1 PST 1 1 QST 1 1 1 1 1SST 1 TST 1 1 1 CSE CSP 1 DSE DSG 1 DSP ESE ESP GSE GSP HSE MSE NSE NSP1 PSE PSP SSE SSP TSP The first column contains the specific sequence,the second column the number of medium DRB1 binding epitopes present inthe sequence fragment, and the subsequent columns describe thespecificity of these epitopes. Allotypes for which no epitopes werefound in any of these sequence fragments were not included in the table.

The lowest epitope content found in the study was within sequencevariants which bind with medium strength to two different DRB1 allotypes(GST, MST, CSE, DSE, DSP, ESP, GSP, HSE, NSE, PSP and SSE). A negativeselection for mutations that:

-   -   substitute any positions to cysteine,    -   change the final threonine to proline, or    -   replace the initial asparagines residue by an aliphatic side        chain, lead to the selection of the following preferred        candidates: GST, NSE, DSE, HSE and SSE.

To make the constructs for the expression of deglycosylated 2F8-HG, theGST and NSE mutations as identified by the above-described analysis wereintroduced into pTomG42F8HG (described in WO 2007059782) usingsite-directed mutagenesis. The constructs were expressed transiently andbinding was determined in the absence and presence of polyclonal humanIgG (Intravenous Immunoglobulin, IVIG, Sanquin Netherlands) (asdescribed in Example 57 of WO 2007059782).

FIG. 8 shows that the binding curves of 2F8-HG-GST and 2F8-HG-NSE in theabsence and presence of IVIG were identical to the binding curve of2F8-HG in the absence and presence of IVIG, respectively. This isconsistent with the hypothesis that deglycosylation does not effect thebinding affinity of the HG-molecules or sensitivity to IVIG.

Example 10 Biochemical Analysis of Non-Glycosylation Mutants of 2F8-HG

Absence of glycosylation in the glycosylation site mutants of 2F8-HG wasconfirmed using High pH Anion Exchange Chromatography—Pulse AmperometricDetection (HPAEC-PAD).

To investigate the monomeric or dimeric configuration of the mutated HGmolecules, a specialized mass spectrometry method was employed topreserve non-covalent interactions between molecules.

HG mutant samples were prepared in aqueous 50 mM ammonium acetatesolutions and introduced into an LC-T nano-electrospray ionizationorthogonal time-of-flight mass spectrometer (Micromass, Manchester, UK),operating in positive ion mode. Source pressure conditions in the LC-Tmass spectrometer and nano-electrospray voltages were optimized foroptimal transmission, the pressure in the interface region was adjustedby reducing the pumping capacity of the rotary pump by closing the valve(Pirani Pressure 6.67e0 mbar).

Spraying conditions were as follows: needle voltage 1275 V, cone voltage200 V, and source temperature 80° C. Borosilicate glass capillaries(Kwik-FiI™, World Precision Instruments Inc., Sarasota, Fla.) were usedon a P-97 puller (Sutter Instrument Co., Novato, Calif.) to prepare thenano-electrospray needles. They were subsequently coated with a thingold layer using an Edwards Scancoat six Pirani 501 sputter coater(Edwards High Vacuum International, Crawley, UK).

FIG. 9 shows a summary of the monomer/dimer ratios obtained for each HGmutant using non-covalent nano-electrospray mass spectrometry at 1 μMprotein concentrations. In agreement with the observations described inExample 54 of WO2007059782, the data indicate that in the absence ofpolyclonal human IgG, 2F8-HG may behave as a bivalent antibody.

Under these experimental conditions, non-glycosylation mutants exhibitedthe same monomer/dimer ratio as 2F8-HG (WT).

Example 11 Functional analysis of non-Glycosylation Mutants of 2F8-HG

Non-glycosylation HG mutants 2F8-HG-GST, 2F8-HG-NSE, 2F8-HG-DSE,2F8-HG-HSE, and 2F8-HG-SSE were shown to bind EGFr with apparentaffinities similar to 2F8-HG (WT) in a binding ELISA, using EGFr proteinas coat (see above). The potency of non-glycosylation 2F8-HG mutants toinhibit ligand-induced EGFr phosphorylation in cells in vitro wascompared to that of 2F8-HG (WT) and 2F8-Fab fragments in thePhosphorylation Inhibition Assay (PIA) as described in example 54 ofWO2007059782. FIG. 10 shows that the potency of non-glycosylation HGmutants to inhibit EGF-induced phosphorylation of EGFr in vitro wassimilar to that of 2F8-HG (WT).

Example 12 Pharmacokinetic Evaluation of Non-Glycosylation Mutants

Pharmacokinetic characteristics of non-glycosylation mutant 2F8-HG-GSTand 2F8-HG-NSE were analyzed in SCID mice supplemented with 0.1 mg7D8-IgG1 as internal control. Pharmacokinetic analysis is explained indetail in example 50 of WO2007059782. Internal control 7D8-IgG1exhibited an equal clearance rate in all mice investigated and wascomparable to the clearance rate of 2F8-IgG4.

FIG. 11 shows that absence of glycosylation of 2F8-HG did not affectplasma clearance.

Example 13 Generation of IgG1 and IgG4 Antibodies with Hinge Regionand/or CH3 Domain Mutations

To investigate the structural requirements for Fab arm exchange, fiveIgG1 mutants were made: an IgG1 with an IgG4 core-hinge (IgG1-P228S)(corresponds to 111 in SEQ ID NO:7), two CH3 domain swap mutants(IgG1-CH3(γ4) and IgG1-P228S—CH3(γ4)), one CH3 point mutant in whichlysine present at position 409 of IgG1 (within the CH3 domain)(corresponds to 292 in SEQ ID NO:7) is replaced for arginine(IgG1-K409R), and one IgG1 with an IgG4 core hinge and K409R mutation(IgG1-P228S-K409R) (FIG. 12). These mutants were made with either Bet v1 or Fel d 1 specificity. Please see WO 2008/119353 (Genmab A(S),especially the examples, for a further description of production ofantibody mutants as well as the Bet v 1 and Fel d 1 specificities.

Two IgG4 mutants were made: one CH3 point mutant in which argininepresent at position 409 of IgG4 (within the CH3 domain) (corresponds to289 in SEQ ID NO:2) is replaced for lysine (IgG4-R409K), and one CH3swap mutant (IgG4-CH3(γ1)) (FIG. 12). These mutants were also made witheither Bet v 1 or Fel d 1 specificity.

Site directed mutagenesis was used to introduce a P228S mutation in thehinge of IgG1 using pEE-G1-wt a Bet v 1 as a template. Quickchangesite-directed mutagenesis kit (Stratagene) was used to create thepEE-G1-CPSC mutant. The polymerase chain reaction (PCR) mix consisted of5 μl pEE-G1a Betv1 DNA template (˜35 ng), 1.5 μl mutagenicprimer-forward (˜150 ng), 1.5 μl mutagenic primer-reverse (˜150 ng), 1μl dNTP mix, 5 μl reaction buffer (10×), 36 μl H2O and finally 1 μl PfuTurbo DNA polymerase. Then the mix was applied to the PCR: 30″ 95° C.,30″ 95° C. (denaturating), 1′ 55° C. (annealing) and 17 minutes 68° C.(elongating). This cycle was repeated 20 times.

DNA digesting and ligation was used to create CH3 domain swap mutantconstructs IgG1-CH3(γ4) and IgG1-P228S-CH3(γ4). Digestion reactions toobtain CH3 domains and vectors without CH3 domains were as follows:˜1500 ng DNA (pEE-G1-betv1, pEE-G1-CPSC and pEE-G4-betv1), 2 μl BSA, 2μl Neb3 buffer, 1 μl SalI and H₂O added to a volume of 20 μl. Incubationat 37° C. for 30′. DNA was purified and eluted with 30 μl H₂O before 1μl SanDI and 3 μl universal buffer was added and incubated at 37° C. for30′. Fragments were subjected to gel electrophoresis on 1% agarose gelswith ethidium bromide. Fragments were cut from the gel under ultravioletlight and dissolved using a DNA purification kit (Amersham). ThepEE-G4-wt SalI/SanDI (which contained IgG4 CH3 domain) fragment wasligated into pEE-G1-wt and pEE-G1-CPSC using following procedure: 1 μltemplate DNA (SalI/SanDI digested pEE-G1-wt and pEE-G1-CPSC), 5 μlSalI/SanDI insert, 4 μl Ligate-it buffer, 9 μl H2O and 1 μl ligase in atotal volume of 20 μl. Ligation was stopped after 5′.

DNA digestion (using ApaI and HindIII) and ligation was used to replacethe VH domain of the bet v 1 mutant antibodies with that ofpEE-G4-a-feld1 wt, following a similar procedure as above.

Site-directed mutagenesis was used to introduce point mutations (K409Ror R409K) into the pEE-γ4 wt, pEE-γ1 and PEE-γ1-P228S constructs.Site-directed mutagenesis was performed using the QuickChange II XLSite-Directed Mutagenesis Kit (Stratagene, Amsterdam, The Netherlands)according to the manufacturer's instructions, with changes as indicatedbelow to increase mutagenic efficiency. This method included theintroduction of a silent extra AccI site to screen for successfulmutagenesis. First, a prePCR mix was used containing 3 μl 10× pfureaction buffer, 1 μl dNTP mix (10 mM), 275 ng forward or reverseprimer, 50 ng template DNA and 0.75 μl Pfu turbo hotstart polymerase. AprePCR was run using a GeneAmp PCR system 9700 (Applied Biosystems):initial denaturation at 94° C. for 5 min; 4 cycles of 94° C. for 30 sec,50° C. for 1 min and 68° C. for 14 min. 25 μl of forward primercontaining prePCR mix was added to 25 μl of reverse primer containingprePCR mix. 0.5 μl Pfu turbo hotstart was added and amplification wasperformed: denaturing at 94° C. for 1 min; 14 cycles of 94° C. for 1min, 50° C. for 1 min and 68° C. for 8 min; 12 cycles of 94° C. for 30sec, 55° C. for 1 min and 68° C. for 8 min.

PCR mixtures were stored at 4° C. until further processing. Next, PCRmixtures were incubated with 1 μl DpnI for 60 min at 37° C. and storedat 4° C. until further processing. 2 μl of the digested PCR products wastransformed in One Shot DNH5α T1^(R) competent E. coli cells(Invitrogen, Breda, The Netherlands) according to the manufacturer'sinstructions (Invitrogen). Next, cells were plated on Luria-Bertani (LB)agar plates containing 50 μg/ml ampicillin. Plates were incubated for16-18 hours at 37° C. until bacterial colonies became evident. Afterscreening by colony PCR and AccI digestion to check for successfulmutagenesis, plasmid was isolated from the bacteria and the mutation wasconfirmed by DNA sequencing. To check if no unwanted extra mutationswere introduced the whole HC coding region was sequenced and did notcontain any additional mutations.

Recombinant antibodies from these constructs were transiently expressedin HEK 293 cells in 3 ml, 6-wells plates (NUNC) or in 125 or 250erlenmeyers (Corning) with 293 Fectin (Invitrogen) as transfectionreagent.

Example 14 Fab Arm Exchange of IgG1 and IgG4 Hinge Region or CH3 DomainMutants

Antibodies were mixed and subsequently incubated with reducedglutathione (GSH) to investigate the exchange of half molecules. GSH(Sigma-Aldrich, St. Louis, Mo.) was dissolved in water before use.

The exchange of half molecules was evaluated by incubating an antibodymixture consisting of Bet v 1 specific antibody (200 ng) and Fel d 1specific antibody (200 ng) in PBS/Azide containing GSH (1 or 10 mM) at37° C. Total incubation volume was 50 μl. After 24 hours samples weredrawn from the incubation mixture in PBS-AT (PBS supplemented with 0.3%bovine serum albumin, 0.1% Tween-20 and 0.05% (w/v) NaN₃). For samplescontaining 10 mM GSH an equimolar amount of iodine-acetamide, a stronglyalkylating agent that inhibits the GSH activity, was added. Samples werestored at 4° C. for measuring of antigen binding and bispecific activity

Levels of Bet v 1 binding antibodies were measured in the antigenbinding test. Samples were incubated with 0.75 mg of protein G Sepharose(Amersham Biosciences, Uppsala, Sweden) in 750 μl PBS-IAT (PBS-ATsupplemented with 1 μg/ml IVIg) in the presence of ¹²⁵I-labeled Bet v 1for 24 h. Next, the Sepharose was washed with PBS-T (PBS supplementedwith 0.1% Tween-20 and 0.05% (w/v) NaN₃) and the amount of radioactivitybound relative to the amount of radioactivity added was measured. Theconcentration of Bet v 1 specific IgG was calculated using purified Betv 1 specific antibodies as a standard (range 0-200 ng per test asdetermined by nephelometer).

The concentration of bispecific IgG (i.e. Fel d 1-Bet v 1 cross-linkingactivity) was measured in the heterologous cross-linking assay. In thisassay, a sample was incubated for 24 h with 0.5 mg Sepharose-coupled catextract, in which Fel d 1 antigen is present, in a total volume of 300μl in PBS-IAT. Subsequently, the Sepharose was washed with PBS-T andincubated for 24 h with ¹²⁵I-labeled Bet v 1, after which the Sepharosewas washed with PBS-T and the amount of radioactivity bound relative tothe amount of radioactivity added was measured. The concentration ofbispecific IgG (Fel d 1-Bet v 1) was calculated using the samecalibration curve as used in the Bet v 1 binding test, which wasobtained from purified Bet v 1 binding IgG. Tests were performed usingantibody-containing supernatants in FreeStyle 293 expression medium,GIBCO/Invitrogen Corporation.

The following antibody mixtures were used:

-   -   Betv1-IgG1 wt with Feld1-IgG1 wt (indicated as IgG1 wt in FIG.        13)    -   Betv1-IgG1 P228S with Feld1-IgG1-P2285 (IgG1-P228S in FIG. 13)    -   Betv1-IgG4-CH3(γ1) with Feld1-IgG4-CH3(γ1) (IgG4-CH3(γ1) in FIG.        13)    -   Betv1-IgG4-R409K with Feld1-IgG4-R409K (IgG4-R409K in FIG. 13)    -   Betv1-IgG1-CH3(γ4) with Feld1-IgG1-CH3(γ4) (IgG1-CH3(γ4) in FIG.        13)    -   Betv1-IgG1-K409R with Feld1-IgG1-K409R (IgG1-K409R in FIG. 13)    -   Betv1-IgG4 wt with Feld1-IgG4 wt (IgG4 wt in FIG. 13)    -   Betv1-IgG1-P228S—CH3(γ4) with Feld1-IgG1-P228S—CH3(γ4)        (IgG1-P228S—CH3(γ4) in FIG. 13)    -   Betv1-IgG1-P228S-K409R with Feld1-IgG1-P228S-K409R        (IgG1-P228S-K409R in FIG. 13)

The results (FIG. 13) showed that at 1 mM GSH, half molecule exchangeoccurs between IgG4 wt, IgG1-P228S-K409R or IgG1-P228S-CH3(γ4)antibodies.

Under these conditions, IgG1 wt, IgG1-P228S, IgG4-CH3(γ1), IgG4—R409K,IgG1-CH3(γ4) or IgG1-K409R antibodies showed no or only minimal exchangeof half molecules. At 10 mM GSH, half molecule exchange was also seen inthe reactions containing IgG1-CH3(γ4) or IgG1-K409R antibodies.

Example 15 Additional CH3 Mutations to Stabilize Dimerization ofHingeless IgG4 Antibody Molecules in the Absence of IVIG

Hingeless IgG4 antibody (HG) molecules form dimers by low affinitynon-covalent interactions. WO/2007/059782 describes that thisdimerization process can be inhibited by using HG IgG4 molecules in thepresence of an excess of irrelevant antibodies. WO/2007/059782 describesa hingeless IgG4 anti-EGFR antibody 2F8-HG.

Construction of pHG-2F8: A vector for the expression of the heavy chainof 2F8-HG: The heavy chain cDNA encoding region of 2F8-HG was codonoptimized and cloned in the pEE6.4 vector (Lonza Biologics, Slough, UK).The resulting vector was named pHG-2F8.

Construction of pKappa2F8: A vector for the production of the lightchain of 2F8 antibodies: The VL region encoding antibody 2F8 was codonoptimized and cloned in the pKappa2F2 vector (a vector encoding thecodon optimized cDNA region of antibody 2F2 (described in WO2004035607)in vector pEE12.4 (Lonza)), replacing the 2F2 VL region with the 2F8 VLregion. The resulting vector was named pKappa-2F8.

Hingeless IgG4 anti-EGFR antibody 2F8-HG has been described inWO/2007/059782. The additional mutations given in the Table below wereintroduced into the CH3 region of hingeless IgG4 antibody 2F8-HG bysite-directed mutagenesis.

KABAT indicates amino acid numbering according to Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991).

EU index indicates amino acid numbering according to EU index asoutlined in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). See also FIG. 14 for comparison of numberingmethods.

Numbering of CH3 mutations KABAT EU index G4 SEQ ID NO: 2 436 F405AF285A 436 F405L F285L 440 R409A R289A 440 R409K R289K

To make the constructs for the expression of the CH3 mutants, themutations were introduced into pHG2F8 using site-directed mutagenesis.

The constructs were expressed transiently in HEK-293F cells bycotransfecting the heavy-chain- and light-chain-encoding plasmids andbinding to purified EGFr was determined in the absence and presence of200 μg/ml polyclonal human IgG (Intravenous Immunoglobulin, IVIG,Sanquin Netherlands).

Binding affinities were determined using an ELISA in which purified EGFr(Sigma, St Louis, Mo.) was coated to 96-well Microlon ELISA plates(Greiner, Germany), 50 ng/well. Plates were blocked with PBSsupplemented with 0.05% Tween 20 and 2% chicken serum. Subsequently,samples, serially diluted in a buffer containing 100 μg/ml polyclonalhuman IgG (Intravenous Immunoglobulin, IVIG, Sanquin Netherlands) wereadded and incubated for 1 h at room temperature (RT). Plates weresubsequently incubated with peroxidase-conjugated rabbit-anti-humankappa light chain (DAKO, Glostrup, Denmark) as detecting antibody anddeveloped with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS; Roche, Mannheim, Germany). Absorbance was measured in amicroplate reader (Biotek, Winooski, Vt.) at 405 nm.

FIG. 14 shows that the binding curve of 2F8-HG in the presence of IVIG(thick dotted line with closed boxes) clearly right-shifts with respectto the binding curve of 2F8-HG without IVIG (thick closed line with openboxes). This difference in avidity for the EGFr coat is consistent withthe idea that, in the presence of IVIG, 2F8-HG binds monovalently. Thebinding curves of the tested mutations, 2F8-HG-F405L, 2F8-HG-F405A,2F8-HG-R409A and 2F8-HG-R409KA, become insensitive to the addition ofIVIG and were super-imposable on the bivalent binding curve of 2F8-HG inthe absence of IVIG. These differences in avidity for the EGFr coat areconsistent with the idea that the 2F8-HG-F405L, 2F8-HG-F405A,2F8-HG-R409A and 2F8-HG-R409K mutations stabilize dimerization of the HGmolecules.

Example 16 Additional CH3 Domain Mutations to Stabilize Dimerization ofHuman IgG4 Antibodies

Following the analysis described in Examples 1 and 2, it washypothesized that in human IgG4, mutations relieving the electrostaticstrain between R409 and K370 (indicated with # in the table below) couldpossibly be used to stabilize IgG4 and prevent Fab-arm exchange.Mutations were introduced into the CH3 domains of IgG4-CD20 andIgG4-EGFr by site-directed mutagenesis.

Mutations as given in the Table below were introduced into the CH3domains of IgG4-CD20 and IgG4-EGFr by site-directed mutagenesis.

KABAT indicates amino acid numbering according to Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). EU indexindicates amino acid numbering according to EU index as outlined inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)). See also FIG. 15 for comparison of numbering methods.

Numbering of CH3 mutations KABAT EU index G4 SEQ ID NO: 2 370 Y349DY229D 372 L351K L231K 376 Q355R Q235R 378 E357T E237T 387 S364D S244D393 K370E K250E 393 K370Q K250Q 436 F405A F285A 436 F405L F285L 440R409A R289A 440 R409K R289K 440 R409L R289L 440 R409M R289M 440 R409TR289T 440 R409W R289W 442 T411V T291V 450 E419Q E299Q 476 L445P L325P

IgG1-CD20 and IgG1-EGFr, IgG4-CD20 and IgG4-EGFr, or IgG4-CH3mutant-CD20and IgG4-CH3mutant-EGFr were mixed and incubated with 0.5 mM GSH asdescribed above. Bispecific activity was determined as described inExample 33 of PCT application, WO 2008/119353 (Genmab A/S).

FIG. 16 shows that bispecific anti-EGFr/CD20 antibodies were formed inmixtures of IgG4 antibodies as well as in mixtures of CH3 domain mutantsQ235R, E299Q, L325P, R289A and S244D. No bispecific activity wasmeasured in mixtures of CH3 domain mutants R289K, R289M, R289L, K250Eand K250Q, indicating that these mutations stabilized dimerization ofhuman IgG4 antibodies. For CH3 domain mutants L231K, Y229D, F285A,F285L, R289W and E237T diminished bispecific activity was measured. TheCH3 domain mutant T291V was unique in that it slowed down the exchangereaction, but reached the same level of exchange as wild-type IgG4 after24 hrs.

Example 17 K_(D) Measurements in CH2—CH3 Constructs Based on IgG4 andIgG4 CH3 Mutants

In order to investigate the CH3-CH3 interaction strength of IgG4,his-tagged constructs were designed based on the Fc-domains human IgG4lacking the hinge region to prevent covalent inter-heavy chain disulfidebonds, his-CH2—CH3(G4). Subsequently, variants of these constructscontaining mutations in the CH3-CH3 interface listed below weregenerated by site-directed mutagenesis.

KABAT indicates amino acid numbering according to Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). EU indexindicates amino acid numbering according to EU index as outlined inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)). See also FIG. 15 for comparison of numbering methods.

Numbering of CH3 mutations KABAT EU index G4 SEQ ID NO: 2 370 Y349DY229D 372 L351K L231K 378 E357T E237T 387 S364D S244D 393 K370E K250E393 K370Q K250Q 436 F405A F285A 436 F405L F285L 440 R409A R289A 440R409K R289K 440 R409L R289L 440 R409M R289M 440 R409W R289W

The monomer/dimer configuration of the his-CH2—CH3(G4) and CH3 mutantswas investigated at different concentrations, ranging from 0.01-10 μMusing non-covalent nano-electrospray mass spectrometry as described inWO2007059782. For his-CH2—CH3(G4) and CH3 mutants signals correspondingto the monomeric (M_(s)) and dimeric (D_(s)) configurations wereintegrated and the relative proportion of each configuration at eachconcentration ([M]₀) was determined as described in example 8.

The K_(D) measured for his-CH2—CH3(G4) (WT) was 4.8×10⁻⁸ M. The relativeK_(D) of each mutant compared to the K_(D) of his-CH2—CH3(G4) (WT) wascalculated and plotted.

FIG. 17 shows that CH3 mutants K250E, K250Q, R289L, R289M and R289K hada lower relative K_(D), which translates into stabilization of theCH3-CH3 interaction compared to his-CH2—CH3(G4) (WT). The S244D mutanthad a K_(D).value, which was comparable to his-CH2—CH3(G4) (WT). The CH3mutants Y229D, L231 K, E237T, F285A, F285L, R289A and R289W showed ahigher relative K_(D), meaning an increase in monomeric behaviorcompared to his-CH2—CH3(G4) (WT).

FIG. 18 shows the correlation between the K_(D) values of the CH3mutants in relation to the % of bispecific activity (after 24 hrscompared to WT IgG4). Mutants that are stabilized do not show bispecificactivity, indicating that Fab-arm exchange does not occur in thesemutants. Mutants that have K_(D) values comparable to WT IgG4 behavesimilar in the generation of bispecific antibodies. FIG. 16 and FIG. 18together show that mutants that have a weaker CH3-CH3 interaction doform bispecific antibodies, but the amount of bispecific antibodies ismuch lower and are not stable over time.

SEQUENCE LISTING

1. A monovalent antibody, which comprises a variable region of aselected antigen specific antibody or an antigen binding part of thesaid region, and (ii) a CH region of an immunoglobulin or a fragmentthereof comprising the CH2 and CH3 regions, wherein the CH region orfragment thereof has been modified such that the region corresponding tothe hinge region and, if the immunoglobulin is not an IgG4 subtype,other regions of the CH region, such as the CH3 region, do not compriseany amino acid residues which are capable of forming disulfide bondswith an identical CH region or other covalent or stable non-covalentinter-heavy chain bonds with an identical CH region in the presence ofpolyclonal human IgG, wherein the antibody is of the IgG4 type and theconstant region of the heavy chain has been modified so that one or moreof the following amino acid substitutions have been made relative thesequence set forth in SEQ ID NO: 4: Tyr (Y) in position 217 has beenreplaced by Arg (R), Leu (L) in position 219 has been replaced by Asn(N) or Gln (Q), Glu (E) in position 225 has been replaced by Thr (T),Val (V) or Ne (I), Ser (5) in position 232 has been replaced by Arg (R)or Lys (K), Thr (T) in position 234 has been replaced by Arg (R), Lys(K) or Asn (N), Leu (L) in position 236 has been replaced by Ser (S) orThr (T), Lys (K) in position 238 has been replaced by Arg (R), Asp (D)in position 267 has been replaced by Thr (T) or Ser (S), Phe (F) inposition 273 has been replaced by Arg (R), Gln (Q), Lys (K) or Tyr (Y),Tyr (Y) in position 275 has been replaced by Gln (Q), Lys (K) or Phe(F), Arg (R) in position 277 has been replaced by Glu (E), Thr (T) inposition 279 has been replaced by Asp (D), Val (V) and Asn (N), or theantibody is of another IgG type and the constant region of the heavychain has been modified so that one or more of the same amino-acidsubstitutions have been made at the positions that correspond to thebefore-mentioned positions for IgG4.
 2. The monovalent antibodyaccording to claim 1, which consists of said variable region and said CHregion.
 3. The monovalent antibody according to claim 1, wherein thevariable region is a VH region.
 4. The monovalent antibody according toclaim 1, wherein the variable region is a VL region.
 5. The monovalentantibody according to claim 1, which does not comprise a CL region. 6.The monovalent antibody according to claim 1, which comprises a heavychain and a light chain, wherein the heavy chain comprises (i) a VHregion of a selected antigen specific antibody or an antigen bindingpart of the said region, and (ii) a CH region as defined in claim 1,with the proviso that the CH region has been modified so that it doesnot comprise any acceptor sites for N-linked glycosylation and the lightchain comprises (i) a VL region of a selected antigen specific antibodyor an antigen binding part of the said region, and (ii) a CL regionwhich, in case of an IgG1 subtype, has been modified such that the CLregion does not contain any amino acids which are capable of formingdisulfide bonds with an identical CL region or other covalent bonds withan identical CL region in the presence of polyclonal human IgG.
 7. Themonovalent antibody according to claim 1, wherein the antibody comprisesa CH1 region.
 8. The monovalent antibody according to claim 1, whereinthe monovalent antibody is an IgG1, IgG2, IgG3, IgG4, IgA or IgDantibody, such as an IgG1, IgG2 or IgG4 antibody.
 9. The monovalentantibody according to claim 1, wherein the monovalent antibody is ahuman antibody.
 10. The monovalent antibody according to claim 1,wherein the monovalent antibody comprises the CH3 region as set forth inSEQ ID NO: 7, but wherein the CH3 region has been modified so that oneor more of the following amino acid substitutions have been made: Arg(R) in position 238 has been replaced by Gin (Q); Asp (D) in position239 has been replaced by Glu (E); Thr (T) in position 249 has beenreplaced by Ala (A); Leu (L) in position 251 has been replaced by Ala(A); Leu (L) in position 251 has been replaced by Val (V); Phe (F) inposition 288 has been replaced by Ala (A); Phe (F) in position 288 hasbeen replaced by Leu (L); Tyr (Y) in position 290 has been replaced byAla (A); Lys (K) in position 292 has been replaced by Arg (R); Lys (K)in position 292 has been replaced by Ala (A); Gln (Q) in position 302has been replaced by Glu (E); and Pro (P) in position 328 has beenreplaced by Leu (L).
 11. The monovalent antibody according to claim 10,wherein Lys (K) in position 292 has been replaced by Arg (R).
 12. Themonovalent antibody according to claim 10, wherein the monovalentantibody further comprises the CH1 and/or CH2 regions as set forth inSEQ ID NO: 7, with the proviso that the CH2 region has been modified sothat it does not comprise any acceptor sites for N-linked glycosylation.13. The monovalent antibody according to claim 6, wherein the monovalentantibody comprises the kappa CL region having the amino acid sequence asset forth in SEQ ID NO: 6, but wherein the sequence has been modified sothat the terminal cysteine residue in position 106 has been replacedwith another amino acid residue or has been deleted.
 14. The monovalentantibody according to claim 6, wherein the monovalent antibody comprisesthe lambda C_(L) region having the amino acid sequence as set forth inSEQ ID NO: 5, but wherein the sequence has been modified so that thecysteine residue in position 104 has been replaced with another aminoacid residue or has been deleted.
 15. The monovalent antibody accordingto claim 10, wherein the monovalent antibody comprises the CH1 region asset forth in SEQ ID NO: 7, but wherein the CH1 region has been modifiedso that Ser (S) in position 14 has been replaced by a cysteine residue.16. The monovalent antibody according to claim 1, wherein the monovalentantibody comprises the CH3 region as set forth in SEQ ID NO: 8, butwherein the CH3 region has been modified so that one or more of the ofthe following amino acid substitutions have been made: Arg (R) inposition 234 has been replaced by Gln (Q); Thr (T) in position 245 hasbeen replaced by Ala (A); Leu (L) in position 247 has been replaced byAla (A); Leu (L) in position 247 has been replaced by Val (V); Met (M)in position 276 has been replaced by Val (V); Phe (F) in position 284has been replaced by Ala (A); Phe (F) in position 284 has been replacedby Leu (L); Tyr (Y) in position 286 has been replaced by Ala (A); Lys(K) in position 288 has been replaced by Arg (R); Lys (K) in position288 has been replaced by Ala (A); Gln (Q) in position 298 has beenreplaced by Glu (E); and Pro (P) in position 324 has been replaced byLeu (L).
 17. The monovalent antibody according to claim 16, wherein Lys(K) in position 288 has been replaced by Arg (R).
 18. The monovalentantibody according to claim 16, wherein the monovalent antibody furthercomprises the CH1 and/or CH2 regions as set forth in SEQ ID NO: 8, withthe proviso that the CH2 region has been modified so that it does notcomprise any acceptor sites for N-linked glycosylation.
 19. Themonovalent antibody according to claim 1, wherein the monovalentantibody comprises the CH3 region as set forth in SEQ ID NO: 9, butwherein the CH3 region has been modified so that one or more of thefollowing amino acid substitutions have been made: Arg (R) in position285 has been replaced by Gln (Q); Thr (T) in position 296 has beenreplaced by Ala (A); Leu (L) in position 298 has been replaced by Ala(A); Leu (L) in position 298 has been replaced by Val (V); Ser (S) inposition 314 has been replaced by Asn (N); Asn (N) in position 322 hasbeen replaced by Lys (K); Met (M) in position 327 has been replaced byVal (V); Phe (F) in position 335 has been replaced by Ala (A); Phe (F)in position 335 has been replaced by Leu (L); Tyr (Y) in position 337has been replaced by Ala (A); Lys (K) in position 339 has been replacedby Arg (R); Lys (K) in position 339 has been replaced by Ala (A); Gln(Q) in position 349 has been replaced by Glu (E); Ne (I) in position 352has been replaced by Val (V); Arg (R) in position 365 has been replacedby His (H); Phe (F) in position 366 has been replaced by Tyr (Y); andPro (P) in position 375 has been replaced by Leu (L), with the provisothat the CH3 region has been modified so that it does not comprise anyacceptor sites for N-linked glycosylation.
 20. The monovalent antibodyaccording to claim 19, wherein Lys (K) in position 339 has been replacedby Arg (R).
 21. The monovalent antibody according to claim 1, whereinthe monovalent antibody further comprises the CH1 and/or CH2 regions asset forth in SEQ ID NO: 9, with the proviso that the CH2 region has beenmodified so that it does not comprise any acceptor sites for N-linkedglycosylation.
 22. The monovalent antibody according to claim 1, whereinthe monovalent antibody comprises the CH3 region as set forth in SEQ IDNO: 4, but wherein the CH3 region has been modified so that one or moreof the following amino acid substitutions have been made: Thr (T) inposition 234 has been replaced by Ala (A); Leu (L) in position 236 hasbeen replaced by Ala (A); Leu (L) in position 236 has been replaced byVal (V); Phe (F) in position 273 has been replaced by Ala (A); Phe (F)in position 273 has been replaced by Leu (L); Tyr (Y) in position 275has been replaced by Ala (A).
 23. The monovalent antibody according toclaim 1, wherein the monovalent antibody, except for the one or moreamino acid substitutions defined in claim 1, comprises the CH3 region asset forth in SEQ ID NO:
 4. 24. The monovalent antibody according toclaim 23, but wherein one or more of the following modifications havebeen made: Glu (E) in position 225 has been replaced by Ala (A); Thr (T)in position 234 has been replaced by Ala (A); Leu (L) in position 236has been replaced by Ala (A); Leu (L) in position 236 has been replacedby Val (V); Leu (L) in position 236 has been replaced by Glu (E); Leu(L) in position 236 has been replaced by Gly (G); Lys (K) in position238 has been replaced by Ala (A); Lys (K) in position 238 has beenreplaced by Ala (A); Asp (D) in position 267 has been replaced by Ala(A); Phe (F) in position 273 has been replaced by Ala (A); Phe (F) inposition 273 has been replaced by Leu (L); Phe (F) in position 273 hasbeen replaced by Asp (D) and/or Tyr (Y) in position 275 has beenreplaced by Glu (E); Phe (F) in position 273 has been replaced by hr (T)and/or Tyr (Y) in position 275 has been replaced by Glu (E); and Tyr (Y)in position 275 has been replaced by Ala (A). 25-36. (canceled)
 37. Themonovalent antibody according to claim 12, wherein the monovalentantibody further comprises the CH2 region as set forth in SEQ ID NO: 4,but wherein one or more of the following modifications have been made:Thr (T) in position 118 has been replaced by Gln (Q) and/or Met (M) inposition 296 has been replaced by Leu (L); two or all three of thefollowing substitutions have been made: Met (M) in position 120 has beenreplaced by Tyr (Y); Ser (S) in position 122 has been replaced by Thr(T); and Thr (T) in position 124 has been replaced by Glu (E); Asn (N)in position 302 has been replaced by Ala (A); and Asn (N) in position302 has been replaced by Ala (A) and Thr (T) in position 175 has beenreplaced by Ala (A) and Glu (E) in position 248 has been replaced by Ala(A). 38-40. (canceled)
 41. The monovalent antibody according to claim 1,wherein the CH region has been modified such that all cysteine residueshave been deleted or substituted with other amino acid residues.
 42. Themonovalent antibody according to claim 41, wherein the CH region hasbeen modified such that the cysteine residues of the hinge region havebeen substituted with amino acid residues that have an uncharged polarside chain or a nonpolar side chain.
 43. (canceled)
 44. The monovalentantibody according to claim 1, which is a human IgG4, wherein one of theamino acid residues corresponding to amino acid residues 106 and 109 ofthe sequence of SEQ ID No: 2 has been substituted with an amino acidresidue different from cysteine, and the other of the amino acidresidues corresponding to amino acid residues 106 and 109 of thesequence of SEQ ID No: 2 has been deleted.
 45. The monovalent antibodyaccording to claim 1, which is a human IgG4, wherein at least the aminoacid residues corresponding to amino acid residues 106 to 109 of the CHsequence of SEQ ID No: 2 have been deleted.
 46. The monovalent antibodyaccording to claim 1, which is a human IgG4, wherein at least the aminoacid residues corresponding to amino acid residues 99 to 110 of thesequence of SEQ ID No: 2 have been deleted.
 47. The monovalent antibodyaccording to claim 1, wherein the CH region, except for any mutationsspecified in claim 1, comprises the amino acid sequence of SEQ ID No: 4,with the proviso that the CH2 region has been modified so that it doesnot comprise any acceptor sites for N-linked glycosylation.
 48. Themonovalent antibody according to claim 1, which is a human IgG4, whereinthe CH region has been modified such that the entire hinge region hasbeen deleted.
 49. The monovalent antibody according to claim 1, whereinthe antibody is of the IgG4 type and the constant region of the heavychain has been modified so that one or more of the followingcombinations of amino acid substitutions have been made relative thesequence set forth in SEQ ID NO: 4: Asp (D) in position 267 has beenreplaced by Ser (S) and Tyr (Y) in position 275 has been replaced by Gln(Q) or Lys (K), Arg (R), Asp (D) in position 267 has been replaced byThr (T) and Tyr (Y) in position 275 has been replaced by Gln (Q) or Lys(K), Arg (R), or the antibody is of another IgG type and the constantregion of the heavy chain has been modified so that the samecombinations of amino-acid substitutions have been made at the positionsthat correspond to the before-mentioned positions for IgG4.
 50. Themonovalent antibody according to claim 1, wherein the sequence of theantibody has been modified so that it does not comprise any acceptorsites for N-linked glycosylation.
 51. The monovalent antibody accordingclaim 50, wherein the NST acceptor site for N-linked glycosylation inthe CH2 region has been modified to a sequence selected from the groupconsisting of: GST, MST, CSE, DSE, DSP, ESP, GSP, HSE, NSE, PSP and SSE.52. The monovalent antibody according to claim 1, which has one or moreof the following characteristics: has a plasma concentration above 10μg/ml for more than 7 days when administered in vivo to a human being orto a SCID mouse at a dosage of 4 mg per kg; has a plasma clearance, asdetermined by the method disclosed in Example 52, which is more than 10times slower than the plasma clearance of a F(ab′)² fragment which hasthe same variable region as the monovalent antibody; and has a serumhalf-life of at least 5 days, such as of at least 14 days, for exampleof from 5 and up to 21 days when administered in vivo to a human beingor a SCID mouse. 53-54. (canceled)
 55. The monovalent antibody accordingclaim 1, preceding claims, wherein the monovalent antibody binds to atarget with a dissociation constant (k_(d)) of 10⁻⁷ M or less, such as10⁻⁸ M or less, which target is selected from: erythropoietin,beta-amyloid, thrombopoietin, interferon-alpha (2a and 2b), -beta (1 b),-gamma, TNFR (CD120a), TNFR II (CD120b), IL-1 R type 1 (CD121 a), IL-1 Rtype 2 (CD121 b), IL-2, IL2R (CD25), IL-2R-beta (CD123), IL-3, IL-4,IL-3R (CD123), IL-4R (CD124), IL-5R (CD125), IL-6R-alpha (CD126), -Beta(CD130), IL-10, IL-1 1, IL-15BP, IL-15R, IL-20, IL-21, TCR variablechain, RANK, RANK-L, CTLA4, CXCR4R, CCR5R, TGF-beta1, -βeta2, -βeta3,G-CSF, GM-CSF, MIF-R (CD74), M-CSF—R (CD1 15), GM-CSFB. (CD1 16),soluble FcRI, sFcRII, sFcRIII, FeRn, Factor VII, Factor VIII, Factor IX,VEGF, VEGFxxx.b, anti-psychotic drugs, anti-depressant drugs,anti-Parkinson drugs, anti-seizure agents, neuromuscular blocking drugs,anti-epileptic drugs, adrenocorticosteroids, insulin, proteins orenzymes involved in regulation of insulin, incretins (GIP and GLP-1) ordrugs mimicking incretin action such as Exenatide and sitagliptin,thyroid hormones, growth hormone, ACTH, oestrogen, testosterone,anti-diuretic hormone, diuretics, blood products such as heparin andEPO, beta-blocking agents, cytotoxic agents, anti-viral drugs,anti-bacterial agents, anti-fungal agents, anti-parasitic drugs,anti-coagulation drugs, anti-inflammatory drugs, anti-asthma drugs,anti-COPD drugs, Viagra, opiates, morphine, vitamins (such as vitamin Cfor conservation), hormones involved in pregnancy such as LH and FSH,hormones involved in sex changes, anti-conceptives and antibodies; VEGF,c-Met, CD20, CD38, IL-8, CD25, CD74, FcalphaRI, FcepsilonRI, acetylcholine receptor, fas, fast, TRAIL, hepatitis virus, hepatitis C virus,envelope E2 of hepatitis C virus, tissue factor, a complex of tissuefactor and Factor VII, EGFr, CD4, and CD28.
 56. (canceled)
 57. Themonovalent antibody according to claim 1, which is conjugated to atherapeutic moiety, such as a cytotoxin, a chemotherapeutic drug, animmunosuppressant or a radioisotope.
 58. A pharmaceutical compositioncomprising a monovalent antibody according to claim 1 and one or morepharmaceutically acceptable excipients, diluents or carriers. 59-60.(canceled)
 61. The method of claim 70, wherein a) the disease ordisorder is cancer, an inflammatory condition or an autoimmune disorder;b) the disease or disorder is a disorder involving undesiredangiogenesis; c) the disease or disorder is treatable by administrationof an antibody against a certain target, wherein the involvement ofimmune system-mediated activities is not necessary or is undesirable forachieving the effects of the administration of the antibody, and whereinsaid antibody specifically binds said antigen; d) the disease ordisorder is treatable by blocking or inhibiting a soluble antigen,wherein multimerization of said antigen may form undesirable immunecomplexes, and wherein said antibody specifically binds said antigen; e)the disease or disorder is treatable by blocking or inhibiting a cellmembrane bound receptor, wherein said receptor may be activated bydimerization of said receptor, and wherein said antibody specificallybinds said receptor. 62-67. (canceled)
 68. A diagnostic agent comprisingthe monovalent antibody according to claim
 1. 69. A nucleic acidconstruct encoding the monovalent antibody according to claim
 1. 70. Amethod of treating a disease or disorder, wherein said method comprisesadministering to a subject in need of such treatment a therapeuticallyeffective amount of a monovalent antibody according to claim 1, or apharmaceutical composition thereof or a nucleic acid construct encodingthe monovalent antibody according to claim
 1. 71. (canceled)
 72. Amethod of preparing a monovalent antibody according to claim 1comprising culturing a host cell comprising a nucleic acid constructencoding the monovalent antibody, so that the monovalent antibody isproduced, and recovering the said monovalent antibody from the cellculture.
 73. (canceled)
 74. A stabilized IgG4 antibody, comprising aheavy chain and a light chain, wherein said heavy chain comprises ahuman IgG4 constant region having the sequence set forth in SEQ. IDNO:2, wherein Lys (K) in position 250 has been replaced by Gln (Q) orGlu (E); and wherein the antibody optionally comprises one or morefurther substitutions, deletions and/or insertions in the constantregion as set forth in SEQ ID NO:2
 75. The stabilized IgG4 antibody ofclaim 74, wherein the antibody does not comprise a Cys-Pro-Pro-Cyssequence in the hinge region.
 76. The stabilized IgG4 antibody of claim75, wherein the CH3 region of the antibody has been replaced by the CH3region of human IgG1, of human IgG2 or of human IgG3.
 77. The stabilizedIgG4 antibody of claim 74, wherein said antibody does not comprise asubstitution of the Leu (L) residue at the position corresponding to 115by a Glu (E).
 78. The stabilized IgG4 antibody of claim 74, wherein saidantibody does comprise a substitution of the Leu (L) residue at theposition corresponding to 115 by a Glu (E).
 79. The stabilized IgG4antibody of claim 74, wherein said antibody comprises one or more of thefollowing substitutions an Ala (A) at position 114, an Ala (A) atposition 116, an Ala (A) at position 117, an Ala (A) at position 177, anAla (A) or Val (V) at position 198 an Ala (A) at position 200, an Ala(A) or Gln (Q) at position
 202. 80. The stabilized IgG4 antibody ofclaim 74, wherein said antibody comprises a CXPC or CPXC sequence in thehinge region, wherein X can be any amino acid except for Pro (P). 81.The stabilized IgG4 antibody of claim 74, wherein said antibody does notcomprise an extended IgG3-like hinge region.
 82. The stabilized IgG4antibody of claim 74, wherein said antibody comprises a CPSC sequence inthe hinge region.
 83. The stabilized IgG4 antibody of claim 74, whereinsaid antibody has less than 25, such as less than 10, e.g. less than 9,8, 7, 6, 5, 4, 3, or 2 substitutions, deletions and/or insertions in theconstant region as set forth in SEQ ID NO:2.
 84. The stabilized IgG4antibody of claim 74, wherein said antibody is less efficient inmediating CDC and/or ADCC than a corresponding IgG1 or IgG3 antibodyhaving the same variable regions.
 85. The stabilized IgG4 antibody ofclaim 74, wherein said antibody is selected from the group consisting ofa human monoclonal antibody, a humanized monoclonal antibody and achimeric monoclonal antibody.
 86. The stabilized IgG4 antibody of claim74, wherein said antibody comprises a human kappa light chain or whereinsaid antibody comprises a human lambda light chain.
 87. (canceled) 88.The stabilized IgG4 antibody of claim 74, wherein said antibody is abivalent antibody or a full-length antibody.
 89. (canceled)
 90. Thestabilized IgG4 antibody of claim 74, wherein said antibody is linked toa compound selected from the group consisting of a cytotoxic agent; aradioisotope; a prodrug or drug, such as a taxane; a cytokine; and achemokine.
 91. The stabilized IgG4 antibody of claim 74, wherein theantibody binds to an antigen selected from the group consisting oferythropoietin, beta-amyloid, thrombopoietin, interferon-alpha (2a and2b), interferon-beta (1b), interferon-gamma, TNFR I (CD120a), TNFR II(CD120b), IL-1 R type 1 (CD121 a), IL-1 R type 2 (CD121 b), IL-2, IL2R(CD25), IL-2R-beta (CD123), IL-3, IL-4, IL-3R (CD123), IL-4R (CD124),IL-5R (CD125), IL-6R-alpha (CD126), -beta (CD130), IL-8, IL-10, IL-1 1,IL-15, IL-15BP, IL-15R, IL-20, IL-21, TCR variable chain, RANK, RANK-L,CTLA4, CXCR4R, CCR5R, TGF-beta1, -beta2, -beta3, G-CSF, GM-CSF, MIF-R(CD74), M-CSF—R (CD115), GM-CSFB (CD116), soluble FcRI, sFcRII, sFcRIII,FcRn, Factor VII, Factor VIII, Factor IX, VEGF, VEGFxxxb, alpha-4integrin, Cd11a, CD18, CD20, CD38, CD25, CD74, FcalphaRI, FcepsilonRI,acetyl choline receptor, fas, fasL, TRAIL, hepatitis virus, hepatitis Cvirus, envelope E2 of hepatitis C virus, tissue factor, a complex oftissue factor and Factor VII, EGFr, CD4, CD28, VLA-1, 2, 3, or 4, LFA-1,MAC-1, l-selectin, PSGL-1, P-selectin, periostin, CD33 (Siglec 3),Siglec 8, TNF, CCU., CCL2, CCL3, CCL4, CCL5, CCL11, CCL13, CCL17, CCL18,CCL20, CCL22, CCL26, CCL27, CX3CL1, LIGHT, EGF, VEGF, TGFalpha, HGF,PDGF, NGF, complement or a related components such as: C1q, C4, C2, C3,C5, C6, C7, C8, C9, MBL, factor B, a Matrix Metallo Protease such as anyof MMP1 to MMP28, CD32b, CD200, CD200R, Killer Immunoglobulin-LikeReceptors (KIRs), NKG2D and related molecules, leukocyte-associatedimmunoglobulin-like receptors (LAIRS), Iy49, PD-L2, CD26, BST-2, ML-IAP(melanoma inhibitor of apoptosis protein), cathepsin D, CD40, CD40R,CD86, a B cell receptor, CD79, PD-1 and a T cell receptor.